Protein synthesis in sperm: dialog between mitochondria and cytoplasm.

PubMed

Gur, Yael; Breitbart, Haim

2008-01-30

Ejaculated sperm are capable of using mRNAs transcripts for protein translation during the final maturation steps before fertilization. In a capacitation-dependent process, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes while the cytoplasmic translation machinery is not involved. Our findings suggest that new proteins are synthesized to replace degraded proteins while swimming and waiting in the female reproductive tract before fertilization, or produced due to the specific needs of the capacitating spermatozoa. In addition, a growing number of articles have reported evidence for the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. It is known that all of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment is far out of proportion to the number of proteins involved, as there have been multiple movements and duplications of genes. However, the evolutionary retention (or secondary uptake) of the mitochondrial machinery for translation of nuclear-encoded mRNAs may shed light on this paradox.

In vivo expression of Salmonella enterica serotype Typhi genes in the blood of patients with typhoid fever in Bangladesh.

PubMed

Sheikh, Alaullah; Charles, Richelle C; Sharmeen, Nusrat; Rollins, Sean M; Harris, Jason B; Bhuiyan, Md Saruar; Arifuzzaman, Mohammad; Khanam, Farhana; Bukka, Archana; Kalsy, Anuj; Porwollik, Steffen; Leung, Daniel T; Brooks, W Abdullah; LaRocque, Regina C; Hohmann, Elizabeth L; Cravioto, Alejandro; Logvinenko, Tanya; Calderwood, Stephen B; McClelland, Michael; Graham, James E; Qadri, Firdausi; Ryan, Edward T

2011-12-01

Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans. We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR. We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.

Regulated capture by exosomes of mRNAs for cytoplasmic tRNA synthetases.

PubMed

Wang, Feng; Xu, Zhiwen; Zhou, Jie; Lo, Wing-Sze; Lau, Ching-Fun; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2013-10-11

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

PubMed Central

Akagi, H; Patton, D E; Miledi, R

1989-01-01

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

PubMed

Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-05-01

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.

PubMed Central

Spindel, E R; Chin, W W; Price, J; Rees, L H; Besser, G M; Habener, J F

1984-01-01

We have prepared and cloned cDNAs derived from poly(A)+ RNA from a human pulmonary carcinoid tumor rich in immunoreactivity to gastrin-releasing peptide, a peptide closely related in structure to amphibian bombesin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to amphibian bombesin were used as hybridization probes to screen a cDNA library prepared from the tumor RNA. Sequencing of the recombinant plasmids shows that human gastrin-releasing peptide (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. RNA blot analyses of tumor RNA show a major mRNA of 900 bases and a minor mRNA of 850 bases. Blot hybridization analyses using human genomic DNA are consistent with a single hGRP-encoding gene. The presence of two mRNAs encoding the hGRP precursor protein in the face of a single hGRP gene raises the possibility of alternative processing of the single RNA transcript. Images PMID:6207529

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

PubMed Central

Damgaard, Christian Kroun; Lykke-Andersen, Jens

2011-01-01

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5′-terminal oligopyrimidine tracts (5′TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5′TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5′TOP mRNA regulation, which upon amino acid starvation assemble onto the 5′ end of 5′TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5′TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5′TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment. PMID:21979918

Glycyrrhizin and glycyrrhetinic acid inhibits alpha-naphthyl isothiocyanate-induced liver injury and bile acid cycle disruption.

PubMed

Wang, Haina; Fang, Zhong-Ze; Meng, Ran; Cao, Yun-Feng; Tanaka, Naoki; Krausz, Kristopher W; Gonzalez, Frank J

2017-07-01

Alpha-naphthyl isothiocyanate (ANIT) is a common hepatotoxicant experimentally used to reproduce the pathologies of drug-induced liver injury in humans, but the mechanism of its toxicity remains unclear. To determine the metabolic alterations following ANIT exposure, metabolomic analyses was performed by use of liquid chromatography-mass spectrometry. Partial least squares discriminant analysis (PLS-DA) of liver, serum, bile, ileum, and cecum of vehicle- and ANIT-treated mice revealed significant alterations of individual bile acids, including increased tauroursodeoxycholic acid, taurohydrodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid, and decreased ω-, β- and tauro-α/β- murideoxycholic acid, cholic acid, and taurocholic acid in the ANIT-treated groups. In accordance with these changes, ANIT treatment altered the expression of mRNAs encoded by genes responsible for the metabolism and transport of bile acids and cholesterol. Pre-treatment of glycyrrhizin (GL) and glycyrrhetinic acid (GA) prevented ANIT-induced liver damage and reversed the alteration of bile acid metabolites and Cyp7a1, Npc1l1, Mttp, and Acat2 mRNAs encoding bile acid transport and metabolism proteins. These results suggested that GL/GA could prevent drug-induced liver injury and ensuing disruption of bile acid metabolism in humans. Published by Elsevier B.V.

Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE)

PubMed Central

Sharon, Dror; Blackshaw, Seth; Cepko, Constance L.; Dryja, Thaddeus P.

2002-01-01

We used the serial analysis of gene expression (SAGE) technique to catalogue and measure the relative levels of expression of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium (RPE) from one or both of two humans, aged 88 and 44 years. The cone photoreceptor contribution to all transcription in the retina was found to be similar in the macula versus the retinal periphery, whereas the rod contribution was greater in the periphery versus the macula. Genes encoding structural proteins for axons were found to be expressed at higher levels in the macula versus the retinal periphery, probably reflecting the large proportion of ganglion cells in the central retina. In comparison with the younger eye, the peripheral retina of the older eye had a substantially higher proportion of mRNAs from genes encoding proteins involved in iron metabolism or protection against oxidative damage and a substantially lower proportion of mRNAs from genes encoding proteins involved in rod phototransduction. These differences may reflect the difference in age between the two donors or merely interindividual variation. The RPE library had numerous previously unencountered tags, suggesting that this cell type has a large, idiosyncratic repertoire of expressed genes. Comparison of these libraries with 100 reported nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a retinal disease and 53 are RPE-specific genes. We expect that these libraries will serve as a resource for understanding the relative expression levels of genes in the retina and the RPE and for identifying additional disease genes. PMID:11756676

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

PubMed Central

Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

2005-01-01

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection. PMID:16163387

RNA Surveillance: Molecular Approaches in Transcript Quality Control and their Implications in Clinical Diseases

PubMed Central

Moraes, Karen CM

2010-01-01

Production of mature mRNAs that encode functional proteins involves highly complex pathways of synthesis, processing and surveillance. At numerous steps during the maturation process, the mRNA transcript undergoes scrutiny by cellular quality control machinery. This extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possibly deleterious proteins. Recent advances in elucidating the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and have revealed that a variety of human diseases are caused by disturbances in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss and gain of function are relevant, and a considerable number of different diseases exemplify the importance of the mechanistic function of RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, highlighting the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of therapeutic strategies. PMID:19829759

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

PubMed Central

Logemann, Elke; Tavernaro, Annette; Schulz, Wolfgang; Somssich, Imre E.; Hahlbrock, Klaus

2000-01-01

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells. PMID:10677554

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

PubMed

Leitao, Louis; Maoret, Jean-José; Biolley, Jean-Philippe

2007-01-01

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

PubMed

Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

2017-01-01

A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development

PubMed Central

Fox, Catherine A.; Dowdle, Megan E.; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

2017-01-01

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation. PMID:27975270

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

Encoding of contextual fear memory requires de novo proteins in the prelimbic cortex

PubMed Central

Rizzo, Valerio; Touzani, Khalid; Raveendra, Bindu L.; Swarnkar, Supriya; Lora, Joan; Kadakkuzha, Beena M.; Liu, Xin-An; Zhang, Chao; Betel, Doron; Stackman, Robert W.; Puthanveettil, Sathyanarayanan V.

2016-01-01

Background Despite our understanding of the significance of the prefrontal cortex in the consolidation of long-term memories (LTM), its role in the encoding of LTM remains elusive. Here we investigated the role of new protein synthesis in the mouse medial prefrontal cortex (mPFC) in encoding contextual fear memory. Methods Because a change in the association of mRNAs to polyribosomes is an indicator of new protein synthesis, we assessed the changes in polyribosome-associated mRNAs in the mPFC following contextual fear conditioning (CFC) in the mouse. Differential gene expression in mPFC was identified by polyribosome profiling (n = 18). The role of new protein synthesis in mPFC was determined by focal inhibition of protein synthesis (n = 131) and by intra-prelimbic cortex manipulation (n = 56) of Homer 3, a candidate identified from polyribosome profiling. Results We identified several mRNAs that are differentially and temporally recruited to polyribosomes in the mPFC following CFC. Inhibition of protein synthesis in the prelimbic (PL), but not in the anterior cingulate cortex (ACC) region of the mPFC immediately after CFC disrupted encoding of contextual fear memory. Intriguingly, inhibition of new protein synthesis in the PL 6 hours after CFC did not impair encoding. Furthermore, expression of Homer 3, an mRNA enriched in polyribosomes following CFC, in the PL constrained encoding of contextual fear memory. Conclusions Our studies identify several molecular substrates of new protein synthesis in the mPFC and establish that encoding of contextual fear memories require new protein synthesis in PL subregion of mPFC. PMID:28503670

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

PubMed Central

Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

2002-01-01

A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.

PubMed

Lyons, Shawn M; Cunningham, Clark H; Welch, Joshua D; Groh, Beezly; Guo, Andrew Y; Wei, Bruce; Whitfield, Michael L; Xiong, Yue; Marzluff, William F

2016-11-02

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

PubMed Central

MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

PubMed

Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods

PubMed Central

Mulindwa, Julius; Leiss, Kevin; Ibberson, David; Kamanyi Marucha, Kevin; Helbig, Claudia; Melo do Nascimento, Larissa; Silvester, Eleanor; Matthews, Keith; Matovu, Enock; Enyaru, John

2018-01-01

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs. PMID:29474390

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

PubMed

Mulindwa, Julius; Leiss, Kevin; Ibberson, David; Kamanyi Marucha, Kevin; Helbig, Claudia; Melo do Nascimento, Larissa; Silvester, Eleanor; Matthews, Keith; Matovu, Enock; Enyaru, John; Clayton, Christine

2018-02-01

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs.

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

PubMed Central

Becker, Jordan T.

2017-01-01

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. PMID:28053097

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

PubMed

Becker, Jordan T; Sherer, Nathan M

2017-03-15

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis -acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins ( gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. Copyright © 2017 American Society for Microbiology.

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Dendritic Glutamate Receptor mRNAs Show Contingent Local Hotspot-Dependent Translational Dynamics

PubMed Central

Kim, Tae Kyung; Sul, Jai-Yoon; Helmfors, Henrik; Langel, Ulo; Kim, Junhyong; Eberwine, James

2014-01-01

SUMMARY Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s-3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite. PMID:24075992

Postnatal changes and sexual dimorphism in collagen expression in mouse skin

PubMed Central

Arai, Koji Y.; Hara, Takuya; Nagatsuka, Toyofumi; Kudo, Chikako; Tsuchiya, Sho; Nomura, Yoshihiro; Nishiyama, Toshio

2017-01-01

To investigate sexual dimorphism and postnatal changes in skin collagen expression, mRNA levels of collagens and their regulatory factors in male and female skin were examined during the first 120 days of age by quantitative realtime PCR. Levels of mRNAs encoding extracellular matrices did not show any differences between male and female mice until day 15. Col1a1 and Col1a2 mRNAs noticeably increased at day 30 and remained at high levels until day 120 in male mice, while those in female mice remained at low levels during the period. Consistent with the mRNA expression, pepsin-soluble type I collagen contents in skin was very high in mature male as compared to female. Col3a1 mRNA in male mice also showed significantly high level at day 120 as compared to female. On the other hand, expression of mRNAs encoding TGF-ßs and their receptors did not show apparent sexual dimorphism although small significant differences were observed at some points. Castration at 60 days of age resulted in a significant decrease in type I collagen mRNA expression within 3 days, and noticeably decreased expression of all fibril collagen mRNAs examined within 14 days, while administration of testosterone tube maintained the mRNA expression at high levels. Despite the in vivo effect of testosterone, administration of physiological concentrations of testosterone did not affect fibril collagen mRNA expression in either human or mouse skin fibroblasts in vitro, suggesting that testosterone does not directly affect collagen expression in fibroblasts. In summary, present study demonstrated dynamic postnatal changes in expression of collagens and their regulatory factors, and suggest that testosterone and its effects on collagen expression are responsible for the skin sexual dimorphism but the effects of testosterone is not due to direct action on dermal fibroblasts. PMID:28494009

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

PubMed

Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

2016-05-17

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Gastric and hepatocellular carcinomas do not overexpress the same ribosomal protein messenger RNAs as colonic carcinoma.

PubMed

Barnard, G F; Staniunas, R J; Mori, M; Puder, M; Jessup, M J; Steele, G D; Chen, L B

1993-09-01

The levels of a number of ribosomal protein mRNAs are reported to be increased in human colon cancer. We have assessed whether selected ribosomal protein mRNAs are overexpressed in other gastrointestinal malignancies, namely gastric and hepatocellular carcinomas. Subtracted complementary DNA libraries were generated from paired samples of human (a) colorectal carcinoma minus adjacent normal colonic mucosa and (b) hepatocellular carcinoma minus adjacent normal liver. Screening of approximately 3% of these library clones determined that ribosomal protein mRNAs encoding L18 and L37 (not previously reported) and P0 and S6 were overexpressed in one or the other library. Their complementary DNA inserts were then used as probes to evaluate their expression in a larger number of paired tumor/normal surgical samples of human colonic, gastric, and hepatocellular carcinomas, by Northern hybridization. The mRNA signal was greater in the colonic carcinoma than in paired adjacent normal colonic mucosa in 38 of 42 cases for P0 [tumor/normal (T/N) ratio = 3.0 +/- 0.3, mean +/- SE, P < 0.001] (G. F. Barnard, R. J. Staniunas, S. Bao, K. Mafune, J. L. Gollan, G. D. Steele, Jr., and L. B. Chen, Cancer Res., 52: 3067-3072, 1992), in 25 of 28 cases for L18 (T/N ratio = 3.7 +/- 0.5, P < 0.001), in 27 of 28 cases for L37 (T/N ratio = 5.3 +/- 0.4, P < 0.001), and in 24 of 28 cases for S6 (T/N ratio = 3.1 +/- 0.5, P < 0.01). The level of mRNA overexpression of L18 and S6 did not correlate with the Dukes' stage of disease. In hepatocellular carcinoma samples, using the same four ribosomal protein complementary DNA probes, only P0 mRNA was significantly increased (T/N ratio = 2.8 +/- 0.4, n = 6, P = 0.047). In gastric carcinoma samples, none of these mRNAs was increased (mean T/N ratios = 0.9-1.2, n = 6). Therefore, gastric and hepatocellular carcinomas do not overexpress the same ribosomal protein mRNAs as do colonic carcinoma.

EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia

PubMed Central

Boczonadi, Veronika; Müller, Juliane S.; Pyle, Angela; Munkley, Jennifer; Dor, Talya; Quartararo, Jade; Ferrero, Ileana; Karcagi, Veronika; Giunta, Michele; Polvikoski, Tuomo; Birchall, Daniel; Princzinger, Agota; Cinnamon, Yuval; Lützkendorf, Susanne; Piko, Henriett; Reza, Mojgan; Florez, Laura; Santibanez-Koref, Mauro; Griffin, Helen; Schuelke, Markus; Elpeleg, Orly; Kalaydjieva, Luba; Lochmüller, Hanns; Elliott, David J.; Chinnery, Patrick F.; Edvardson, Shimon; Horvath, Rita

2014-01-01

The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease. PMID:24989451

IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and Other mRNAs encoding mitochondrial proteins.

PubMed

Dai, Ning; Zhao, Liping; Wrighting, Diedra; Krämer, Dana; Majithia, Amit; Wang, Yanqun; Cracan, Valentin; Borges-Rivera, Diego; Mootha, Vamsi K; Nahrendorf, Matthias; Thorburn, David R; Minichiello, Liliana; Altshuler, David; Avruch, Joseph

2015-04-07

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs. Copyright © 2015 Elsevier Inc. All rights reserved.

Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

PubMed

Kleene, Kenneth C

2016-03-01

Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression

PubMed Central

Fahey, Ciara; Kenny, Elaine M; Terenin, Ilya M; Dmitriev, Sergey E; Cormican, Paul; Morris, Derek W; Shatsky, Ivan N; Baranov, Pavel V

2015-01-01

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. DOI: http://dx.doi.org/10.7554/eLife.03971.001 PMID:25621764

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

PubMed Central

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-01-01

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC. PMID:28685749

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

PubMed

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-07-07

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNA Lys (UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

PubMed Central

Spatuzza, Carmen; Schiavone, Marco; Di Salle, Emanuela; Janda, Elzbieta; Sardiello, Marco; Fiume, Giuseppe; Fierro, Olga; Simonetta, Marco; Argiriou, Notis; Faraonio, Raffaella; Capparelli, Rosanna; Quinto, Ileana

2008-01-01

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk. PMID:18596081

Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus

PubMed Central

Di, Han; Madden, Joseph C.; Morantz, Esther K.; Tang, Hsin-Yao; Graham, Rachel L.; Baric, Ralph S.

2017-01-01

Members of the order Nidovirales express their structural protein ORFs from a nested set of 3′ subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3′ region, but some are in the 5′ ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3′ ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader–body junction sequences. Sg mRNAs encoding E′, GP2, or ORF5a as their 5′ ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses. PMID:29073030

Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms.

PubMed

Minia, Igor; Merce, Clementine; Terrao, Monica; Clayton, Christine

2016-09-01

African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C-41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms.

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grzybowska, Ewa A., E-mail: ewag@coi.waw.pl

2012-07-20

Highlights: Black-Right-Pointing-Pointer Functional characteristics of intronless genes (IGs). Black-Right-Pointing-Pointer Diseases associated with IGs. Black-Right-Pointing-Pointer Origin and evolution of IGs. Black-Right-Pointing-Pointer mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associatedmore » diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.« less

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

Gene Expression Profiling in Pachyonychia Congenita Skin

PubMed Central

Cao, Yu-An; Hickerson, Robyn P.; Seegmiller, Brandon L.; Grapov, Dmitry; Gross, Maren M.; Bessette, Marc R.; Phinney, Brett S.; Flores, Manuel A.; Speaker, Tycho J.; Vermeulen, Annaleen; Bravo, Albert A.; Bruckner, Anna L.; Milstone, Leonard M.; Schwartz, Mary E.; Rice, Robert H.; Kaspar, Roger L.

2015-01-01

Background Pachyonychia congenita (PC) is a skin disorder resulting from mutations in keratin (K) proteins including K6a, K6b, K16, and K17. One of the major symptoms is painful plantar keratoderma. The pathogenic sequelae resulting from the keratin mutations remain unclear. Objective To better understand PC pathogenesis. Methods RNA profiling was performed on biopsies taken from PC-involved and uninvolved plantar skin of seven genotyped PC patients (two K6a, one K6b, three K16, and one K17) as well as from control volunteers. Protein profiling was generated from tape-stripping samples. Results A comparison of PC-involved skin biopsies to adjacent uninvolved plantar skin identified 112 differentially-expressed mRNAs common to patient groups harboring K6 (i.e., both K6a and K6b) and K16 mutations. Among these mRNAs, 25 encode structural proteins including keratins, small proline-rich and late cornified envelope proteins, 20 are related to metabolism and 16 encode proteases, peptidases, and their inhibitors including kallikrein-related peptidases (KLKs), and serine protease inhibitors (SERPINs). mRNAs were also identified to be differentially expressed only in K6 (81) or K16 (141) patient samples. Furthermore, 13 mRNAs were identified that may be involved in pain including nociception and neuropathy. Protein profiling, comparing three K6a plantar tape-stripping samples to non-PC controls, showed changes in the PC corneocytes similar, but not identical, to the mRNA analysis. Conclusion Many differentially-expressed genes identified in PC-involved skin encode components critical for skin barrier homeostasis including keratinocyte proliferation, differentiation, cornification, and desquamation. The profiling data provide a foundation for unraveling the pathogenesis of PC and identifying targets for developing effective PC therapeutics. PMID:25656049

Spliced integrated retrotransposed element (SpIRE) formation in the human genome.

PubMed

Larson, Peter A; Moldovan, John B; Jasti, Naveen; Kidd, Jeffrey M; Beck, Christine R; Moran, John V

2018-03-01

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5' untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5'UTR or 5'UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5'UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5'UTR and 5'UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5'UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5'UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary "dead-ends" in the L1 retrotransposition process, mutations within the L1 5'UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.

Spliced integrated retrotransposed element (SpIRE) formation in the human genome

PubMed Central

Larson, Peter A.; Moldovan, John B.; Jasti, Naveen; Kidd, Jeffrey M.; Beck, Christine R.; Moran, John V.

2018-01-01

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5′ untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5′UTR or 5′UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5′UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5′UTR and 5′UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5′UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5′UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary “dead-ends” in the L1 retrotransposition process, mutations within the L1 5′UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation. PMID:29505568

Translation regulation of mammalian selenoproteins.

PubMed

Vindry, Caroline; Ohlmann, Théophile; Chavatte, Laurent

2018-05-09

Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21 st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins. The insertion of selenocysteine is a non-canonical translational event, based on the recoding of a UGA codon in selenoprotein mRNAs, normally used as a stop codon in other cellular mRNAs. Two RNA molecules and associated partners are crucial components of the selenocysteine insertion machinery, the Sec-tRNA [Ser]Sec devoted to UGA codon recognition and the SECIS elements located in the 3'UTR of selenoprotein mRNAs. The translational UGA recoding event is a limiting stage of selenoprotein expression and its efficiency is regulated by several factors. The control of selenoproteome expression is crucial for redox homeostasis and antioxidant defense of mammalian organisms. In this review, we summarize current knowledge on the co-translational insertion of selenocysteine into selenoproteins, and its layers of regulation. Copyright © 2018. Published by Elsevier B.V.

The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile-X protein ortholog in Drosophila neurons

PubMed Central

Bienkowski, Rick S.; Banerjee, Ayan; Rounds, J. Christopher; Rha, Jennifer; Omotade, Omotola F.; Gross, Christina; Morris, Kevin J.; Leung, Sara W.; Pak, ChangHui; Jones, Stephanie K.; Santoro, Michael R.; Warren, Stephen T.; Zheng, James Q.; Bassell, Gary J.; Corbett, Anita H.; Moberg, Kenneth H.

2017-01-01

Summary The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA-binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with and the Fragile-X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII but not futsch mRNA, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A)-tail length similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. In sum these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14. PMID:28793261

Viral tRNA Mimicry from a Biocommunicative Perspective

PubMed Central

Ariza-Mateos, Ascensión; Gómez, Jordi

2017-01-01

RNA viruses have very small genomes which limits the functions they can encode. One of the strategies employed by these viruses is to mimic key factors of the host cell so they can take advantage of the interactions and activities these factors typically participate in. The viral RNA genome itself was first observed to mimic cellular tRNA over 40 years ago. Since then researchers have confirmed that distinct families of RNA viruses are accessible to a battery of cellular factors involved in tRNA-related activities. Recently, potential tRNA-like structures have been detected within the sequences of a 100 mRNAs taken from human cells, one of these being the host defense interferon-alpha mRNA; these are then additional to the examples found in bacterial and yeast mRNAs. The mimetic relationship between tRNA, cellular mRNA, and viral RNA is the central focus of two considerations described below. These are subsequently used as a preface for a final hypothesis drawing on concepts relating to mimicry from the social sciences and humanities, such as power relations and creativity. Firstly, the presence of tRNA-like structures in mRNAs indicates that the viral tRNA-like signal could be mimicking tRNA-like elements that are contextualized by the specific carrier mRNAs, rather than, or in addition to, the tRNA itself, which would significantly increase the number of potential semiotic relations mediated by the viral signals. Secondly, and in particular, mimicking a host defense mRNA could be considered a potential new viral strategy for survival. Finally, we propose that mRNA’s mimicry of tRNA could be indicative of an ancestral intracellular conflict in which species of mRNAs invaded the cell, but from within. As the meaning of the mimetic signal depends on the context, in this case, the conflict that arises when the viral signal enters the cell can change the meaning of the mRNAs’ internal tRNA-like signals, from their current significance to that they had in the distant past. PMID:29259593

Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA

PubMed Central

Yu, Jia; Russell, J. Eric

2001-01-01

Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway. PMID:11486027

Chromosomal localization and cDNA cloning of the human DBP and TEF genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khatib, Z.A.; Inaba, T.; Valentine, M.

1994-09-15

The authors have isolated cDNA and genomic clones and determined the human chromosome positions of two genes encoding transcription factors expressed in the liver and the pituitary gland: albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). Both proteins have been identified as members of the PAR (proline and acidic amino acid-rich) subfamily of bZIP transcription factors in the rat, but human homologues have not been characterized. Using a fluorescence in situ hybridization technique, the DBP locus was assigned to chromosome 19q13, and TEF to chromosome 22q13. Each assignment was confirmed by means of human chromosome segregation in somatic cellmore » hybrids. Coding sequences of DBP and TEF, extending beyond the bZIP domain to the PAR region, were highly conserved in both human-human and interspecies comparisons. Conservation of the exon-intron boundaries of each bZIP domain-encoding exon suggested derivation from a common ancestral gene. DBP and TEF mRNAs were expressed in all tissues and cell lines examined, including brain, lung, liver, spleen, and kidney. Knowledge of the human chromosome locations of these PAR proteins will facilitate studies to assess their involvement in carcinogenesis and other fundamental biological processes. 37 refs., 5 figs., 1 tab.« less

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

PubMed

Thomsen, Rune; Pallesen, Jonatan; Daugaard, Tina F; Børglum, Anders D; Nielsen, Anders L

2013-11-01

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. Copyright © 2013 Wiley Periodicals, Inc.

Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

PubMed Central

Aligeti, Mounavya; Behrens, Ryan T.; Pocock, Ginger M.; Schindelin, Johannes; Dietz, Christian; Eliceiri, Kevin W.; Swanson, Chad M.; Malim, Michael H.; Ahlquist, Paul

2014-01-01

ABSTRACT Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression. PMID:25275125

nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

PubMed Central

Gandin, Valentina; Masvidal, Laia; Hulea, Laura; Gravel, Simon-Pierre; Cargnello, Marie; McLaughlan, Shannon; Cai, Yutian; Balanathan, Preetika; Morita, Masahiro; Rajakumar, Arjuna; Furic, Luc; Pollak, Michael; Porco, John A.; St-Pierre, Julie; Pelletier, Jerry; Larsson, Ola; Topisirovic, Ivan

2016-01-01

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5′ TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5′ TOP motif but that 5′ UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5′ UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5′ UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5′ UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5′ UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5′ UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells. PMID:26984228

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

PubMed

Uras, Fikriye; Küçük, Burhanettin; Bingöl Özakpınar, Özlem; Demir, Ahmet Muzaffer

2015-03-05

Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Two rat brain staufen isoforms differentially bind RNA.

PubMed

Monshausen, M; Putz, U; Rehbein, M; Schweizer, M; DesGroseillers, L; Kuhl, D; Richter, D; Kindler, S

2001-01-01

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

PubMed

Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery. Copyright © 2017 Pereira et al.

CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate rpL37 5'-terminal oligopyrimidine mRNA metabolism.

PubMed

Schwartz, Elena I; Intine, Robert V; Maraia, Richard J

2004-11-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3' UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5' regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S(366)) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5' untranslated regions comprised of terminal oligopyrimidine (5'TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S(366) represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S(366) phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5'TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A(366) than with La S(366), concomitant with La A(366)-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S(366) in vivo, that this limits 5'TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.

An efficient system for selectively altering genetic information within mRNAs

PubMed Central

Montiel-González, Maria Fernanda; Vallecillo-Viejo, Isabel C.; Rosenthal, Joshua J. C.

2016-01-01

Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a UAG premature termination codon (PTC) to tryptophan (UGG) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool. PMID:27557710

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

PubMed

Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

A New Framework for Understanding IRES-mediated Translation

PubMed Central

Komar, Anton A.; Mazumder, Barsanjit; Merrick, William C.

2012-01-01

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m7G-assisted internal initiation). PMID:22555019

Nonsense-mediated mRNA decay: inter-individual variability and human disease

PubMed Central

Nguyen, Lam Son; Wilkinson, Miles; Gecz, Jozef

2013-01-01

Nonsense-Mediated mRNA Decay (NMD) is a regulatory pathway that functions to degrade transcripts containing premature termination codons (PTCs) and to maintain normal transcriptome homeostasis. Nonsense and frameshift mutations that generate PTCs cause approximately one-third of all known human genetic diseases and thus NMD has a potentially important role in human disease. In genetic disorders in which the affected genes carry PTC-generating mutations, NMD acts as a double-edge sword. While it can benefit the patient by degrading PTC-containing mRNAs that encode detrimental, dominant-negative truncated proteins, it can also make the disease worse when a PTC-containing mRNA is degraded that encodes a mutant but still functional protein. There is evidence that the magnitude of NMD varies between individuals, which, in turn, has been shown to correlate with both clinical presentations and the patients’ responses to drugs that promote read-through of PTCs. In this review, we examine the evidence supporting the existence of inter-individual variability in NMD efficiency and discuss the genetic factors that underlie this variability. We propose that inter-individual variability in NMD efficiency is a common phenomenon in human populations and that an individual’s NMD efficiency should be taken into consideration when testing, developing, and making therapeutic decisions for diseases caused by genes harboring PTCs. PMID:24239855

Differential gene expression in ripening banana fruit.

PubMed Central

Clendennen, S K; May, G D

1997-01-01

During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants. PMID:9342866

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development.

PubMed

Schratt, Gerhard M; Nigh, Elizabeth A; Chen, Wen G; Hu, Linda; Greenberg, Michael E

2004-08-18

Local regulation of mRNA translation plays an important role in axon guidance, synaptic development, and neuronal plasticity. Little is known, however, regarding the mechanisms that control translation in neurons, and only a few mRNAs have been identified that are locally translated within axon and dendrites. Using Affymetrix gene arrays to identify mRNAs that are newly associated with polysomes after exposure to BDNF, we identified subsets of mRNAs for which translation is enhanced in neurons at different developmental stages. In mature neurons, many of these mRNAs encode proteins that are known to function at synapses, including CamKIIalpha, NMDA receptor subunits, and the postsynaptic density (PSD) scaffolding protein Homer2. BDNF regulates the translation of Homer2 locally in the synaptodendritic compartment by activating translational initiation via a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway. These findings suggest that BDNF likely regulates synaptic function by inducing the local synthesis of numerous synaptic proteins. The local translation of the cytoskeleton-associated protein Homer2 in particular might have important implications for growth cone dynamics and dendritic spine development.

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Axonal localization and mitochondrial association of precursor microRNA 338

PubMed Central

Vargas, Jose Norberto S.; Kar, Amar N.; Kowalak, Jeffrey A.; Gale, Jenna R.; Aschrafi, Armaz; Chen, Cai-Yun; Gioio, Anthony E.; Kaplan, Barry B.

2016-01-01

microRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can be locally processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatio-temporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs. PMID:27229124

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

PubMed

Figueiredo, B C; Almazan, G; Ma, Y; Tetzlaff, W; Miller, F D; Cuello, A C

1993-03-01

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

PubMed

Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

2016-01-01

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

Molecular cloning and expression of the CRISP family of proteins in the boar.

PubMed

Vadnais, Melissa L; Foster, Douglas N; Roberts, Kenneth P

2008-12-01

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

PubMed

Calder, Michele D; Watson, Patricia H; Watson, Andrew J

2011-11-01

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonalmore » monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.« less

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

PubMed

Tajaddod, Mansoureh; Tanzer, Andrea; Licht, Konstantin; Wolfinger, Michael T; Badelt, Stefan; Huber, Florian; Pusch, Oliver; Schopoff, Sandy; Janisiw, Michael; Hofacker, Ivo; Jantsch, Michael F

2016-10-25

Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

PubMed Central

Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

2015-01-01

Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

PubMed

Bogerd, H P; Wiegand, H L; Yang, J; Cullen, B R

2000-10-01

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia.

PubMed

Klahre, U; Hemmings-Mieszczak, M; Filipowicz, W

1995-06-01

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3'-untranslated regions (3'-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3' non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event occurring in this intron.

Light-regulated and organ-specific expression of types 1, 2, and 3 light-harvesting complex b mRNAs in Ginkgo biloba.

PubMed Central

Chinn, E; Silverthorne, J; Hohtola, A

1995-01-01

In a prior study (E. Chinn and J. Silverthorne [1993] Plant Physiol 103: 727-732) we showed that the gymnosperm Ginkgo biloba was completely dependent on light for chlorophyll synthesis and chloroplast development and that expression of light-harvesting complex b (Lhcb) mRNAs was substantially increased by light. However, dark-grown seedlings that were transferred to constant white light took significantly longer than angiosperm seedlings to initiate a program of photomorphogenesis and the stems failed to green completely. We have prepared type-specific probes for mRNAs encoding major polypeptides of light-harvesting complex II (Lhcb1, Lhcb2, and Lhcb3) and have used these to analyze the expression of individual Lhcb mRNAs during greening. All three sequences accumulated in the top portions of dark-grown seedlings transferred to light, but, as was seen previously for total Lhcb mRNAs, there was a transient, reproducible decline in the levels of all three mRNAs after 4 d in the light. This transient decrease in Lhcb mRNA levels was not paralleled by a decrease in Chl accumulation. By contrast, there were significantly lower levels of all three Lhcb mRNAs in the lower portions of greening dark-grown stems as well as lower Chl levels. We conclude that although the tops of the plants have the capacity to etiolate and green, Gingko seedling stems continue a program of development into woody tissue in darkness that precludes greening when the seedlings are transferred to the light. PMID:7724674

UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs

PubMed Central

Mignone, Flavio; Grillo, Giorgio; Licciulli, Flavio; Iacono, Michele; Liuni, Sabino; Kersey, Paul J.; Duarte, Jorge; Saccone, Cecilia; Pesole, Graziano

2005-01-01

The 5′ and 3′ untranslated regions of eukaryotic mRNAs play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated (and also collated as the UTRsite database) and cross-links to genomic and protein data are provided. The integration of UTRdb with genomic and protein data has allowed the implementation of a powerful retrieval resource for the selection and extraction of UTR subsets based on their genomic coordinates and/or features of the protein encoded by the relevant mRNA (e.g. GO term, PFAM domain, etc.). All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://www.ba.itb.cnr.it/UTR/. PMID:15608165

Virus-encoded miRNAs in Ebola virus disease.

PubMed

Duy, Janice; Honko, Anna N; Altamura, Louis A; Bixler, Sandra L; Wollen-Roberts, Suzanne; Wauquier, Nadia; O'Hearn, Aileen; Mucker, Eric M; Johnson, Joshua C; Shamblin, Joshua D; Zelko, Justine; Botto, Miriam A; Bangura, James; Coomber, Moinya; Pitt, M Louise; Gonzalez, Jean-Paul; Schoepp, Randal J; Goff, Arthur J; Minogue, Timothy D

2018-04-24

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

PubMed

Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

PubMed Central

Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

The race to decipher the top secrets of TOP mRNAs.

PubMed

Meyuhas, Oded; Kahan, Tamar

2015-07-01

Cells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5' Terminal OligoPyrimidine motif (5'TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action--a repressor or activator. The present chapter critically reviews the development in these three avenues of research with a special emphasis on the two "top secrets" of the TOP mRNA family: the scope of its members and the identity of the proximal cellular regulator(s). This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Genetic and Molecular Characterization of the Caenorhabditis Elegans Spermatogenesis-Defective Gene Spe-17

PubMed Central

L'Hernault, S. W.; Benian, G. M.; Emmons, R. B.

1993-01-01

Two self-sterile mutations that define the spermatogenesis-defective gene spe-17 have been analyzed. These mutations affect unc-22 and fail to complement each other for both Unc-22 and spermatogenesis defects. Both of these mutations are deficiencies (hcDf1 and hDf13) that affect more than one transcription unit. Genomic DNA adjacent to and including the region deleted by the smaller deficiency (hcDf1) has been sequenced and four mRNAs (including unc-22) have been localized to this sequenced region. The three non unc-22 mRNAs are shown to be sex-specific: a 1.2-kb mRNA that can be detected in sperm-free hermaphrodites and 1.2- and 0.56-kb mRNAs found in males. hDf13 deletes at least 55 kb of chromosome IV, including all of unc-22, both male-specific mRNAs and at least part of the female-specific mRNA. hcDf1, which is approximately 15.6 kb, deletes only the 5' end of unc-22 and the gene that encodes the 0.56-kb male-specific mRNA. The common defect that apparently accounts for the defective sperm in hcDf1 and hDf13 homozygotes is deletion of the spe-17 gene, which encodes the 0.56-kb mRNA. Strains carrying two copies of either deletion are self-fertile when they are transgenic for any of four extrachromosomal array that include spe-17. We have sequenced two spe-17 cDNAs, and the deduced 142 amino acid protein sequence is highly charged and rich in serine and threonine, but shows no significant homology to any previously determined protein sequence. PMID:8349108

Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein

PubMed Central

Droll, Dorothea; Minia, Igor; Fadda, Abeer; Singh, Aditi; Stewart, Mhairi; Queiroz, Rafael; Clayton, Christine

2013-01-01

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures. PMID:23592996

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

PubMed Central

Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

2016-01-01

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

Signal sequence-independent targeting of MID2 mRNA to the endoplasmic reticulum by the yeast RNA-binding protein Khd1p.

PubMed

Syed, Muhammad Ibrahim; Moorthy, Balaji T; Jenner, Andreas; Fetka, Ingrid; Jansen, Ralf-Peter

2018-05-17

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA. ER targeting of at least two mRNAs, MID2 and SLG1/WSC1, requires KHD1 but is independent of their translation. Together, our results suggest interdependence of Khd1p and mRNA for their targeting to the ER and presents additional evidence for signal sequence-independent, RBP-mediated mRNA targeting. © 2018 Federation of European Biochemical Societies.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Ribosomes slide on lysine-encoding homopolymeric A stretches

PubMed Central

Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

2015-01-01

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

The RNA Binding Protein Tudor-SN Is Essential for Stress Tolerance and Stabilizes Levels of Stress-Responsive mRNAs Encoding Secreted Proteins in Arabidopsis[C][W][OA

PubMed Central

dit Frey, Nicolas Frei; Muller, Philippe; Jammes, Fabien; Kizis, Dimosthenis; Leung, Jeffrey; Perrot-Rechenmann, Catherine; Bianchi, Michele Wolfe

2010-01-01

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death–associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway. PMID:20484005

MicroRNA signature of the human developing pancreas.

PubMed

Rosero, Samuel; Bravo-Egana, Valia; Jiang, Zhijie; Khuri, Sawsan; Tsinoremas, Nicholas; Klein, Dagmar; Sabates, Eduardo; Correa-Medina, Mayrin; Ricordi, Camillo; Domínguez-Bendala, Juan; Diez, Juan; Pastori, Ricardo L

2010-09-22

MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase algorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.

MicroRNA signature of the human developing pancreas

PubMed Central

2010-01-01

Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas. PMID:20860821

CK2 Is Responsible for Phosphorylation of Human La Protein Serine-366 and Can Modulate rpL37 5′-Terminal Oligopyrimidine mRNA Metabolism

PubMed Central

Schwartz, Elena I.; Intine, Robert V.; Maraia, Richard J.

2004-01-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3′ UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5′ regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S366) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5′ untranslated regions comprised of terminal oligopyrimidine (5′TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S366 represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S366 phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5′TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A366 than with La S366, concomitant with La A366-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S366 in vivo, that this limits 5′TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery. PMID:15485924

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

PubMed Central

Spindel, E R; Gibson, B W; Reeve, J R; Kelly, M

1990-01-01

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach. Images PMID:2263631

Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria.

PubMed

Cahoon, A Bruce; Qureshi, Ali A

2018-06-01

The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3' untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3' UTR upstream of the 5' start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3'-5' junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5' ends (the start AUG) and a variable 3' terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3' ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3' termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3' termini of these mRNAs-adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

PubMed Central

2014-01-01

Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission. PMID:24898526

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

PubMed

Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication

PubMed Central

Courtney, David G.

2018-01-01

Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N6 position (m6A), although the precise location of these m6A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m6A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m6A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m6A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m6A addition on SV40 transcripts and identified two m6A sites on the viral early transcripts and eleven m6A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m6A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m6A residues and our data instead suggest that m6A enhances the translation of viral late transcripts. Together, these data argue that the addition of m6A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication. PMID:29447282

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway.

PubMed

Pende, A; Tremmel, K D; DeMaria, C T; Blaxall, B C; Minobe, W A; Sherman, J A; Bisognano, J D; Bristow, M R; Brewer, G; Port, J

1996-04-05

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

Pre and postprandial changes in orexigenic and anorexigenic factors in channel catfish Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

We examined pre- and postprandial changes in the expression of plasma ghrelin (GHRL) and mRNAs encoding GRLN, cocaine and amphetamine regulated transcript (CART), neuropeptide Y (NPY), and cholecystokinin (CCK) in channel catfish. Fish were either offered feed (Fed) or fasted (Unfed). Feeding incr...

Pre and postprandial changes in orexigenic and anorexigenic factors in channel catfish Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

Ghrelin (GRLN), cocaine and amphetamine regulated transcript (CART), neuropeptide Y (NPY), and cholecystokinin (CCK) are neuropeptides involved in the regulation of appetite and feeding in vertebrates. We examined pre- and postprandial changes in the expression of plasma GHRL and mRNAs encoding GRL...

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

Nuclear mRNA Surveillance Mechanisms: Function and Links to Human Disease.

PubMed

Singh, Pragyan; Saha, Upasana; Paira, Sunirmal; Das, Biswadip

2018-05-11

Production of export-competent mRNAs involves transcription and a series of dynamic processing and modification events of pre-messenger RNAs in the nucleus. Mutations in the genes encoding the transcription and mRNP processing machinery and the complexities involved in the biogenesis events lead to the formation of aberrant messages. These faulty transcripts are promptly eliminated by the nuclear RNA exosome and its cofactors to safeguard the cells and organisms from genetic catastrophe. Mutations in the components of the core nuclear exosome and its cofactors lead to the tissue-specific dysfunction of exosomal activities, which are linked to diverse human diseases and disorders. In this article, we examine the structure and function of both the yeast and human RNA exosome complex and its cofactors, discuss the nature of the various altered amino acid residues implicated in these diseases with the speculative mechanisms of the mutation-induced disorders and project the frontier and prospective avenues of the future research in this field. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glucose-Regulated Phosphorylation of the PUF Protein Puf3 Regulates the Translational Fate of Its Bound mRNAs and Association with RNA Granules.

PubMed

Lee, Chien-Der; Tu, Benjamin P

2015-06-16

PUF proteins are post-transcriptional regulators that bind to the 3' UTRs of mRNA transcripts. Herein, we show how a yeast PUF protein, Puf3p, responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. Upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. Such nutrient-responsive phosphorylation toggles the activity of Puf3p to promote either degradation or translation of these mRNAs according to the needs of the cell. Moreover, activation of translation of pre-existing mRNAs might enable rapid adjustment to environmental changes without the need for de novo transcription. Strikingly, a Puf3p phosphomutant no longer promotes translation but becomes trapped in intracellular foci in an mRNA-dependent manner. Our findings suggest that the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

PubMed Central

Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7.

PubMed

Zhang, Fang; Sodroski, Catherine; Cha, Helen; Li, Qisheng; Liang, T Jake

2017-01-01

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

PubMed

Tappaz, M; Bitoun, M; Reymond, I; Sergeant, A

1999-09-01

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

PubMed

Nydam, Marie L; Netuschil, Nikolai; Sanders, Erin; Langenbacher, Adam; Lewis, Daniel D; Taketa, Daryl A; Marimuthu, Arumugapradeep; Gracey, Andrew Y; De Tomaso, Anthony W

2013-01-01

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

PubMed Central

Loughran, Gary; Jungreis, Irwin; Tzani, Ioanna; Power, Michael; Dmitriev, Ruslan I.; Ivanov, Ivaylo P.; Kellis, Manolis; Atkins, John F.

2018-01-01

Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid–long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor. PMID:29386352

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

PubMed Central

Lee, Myon-Hee; Hook, Brad; Pan, Guangjin; Kershner, Aaron M; Merritt, Christopher; Seydoux, Geraldine; Thomson, James A; Wickens, Marvin; Kimble, Judith

2007-01-01

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression. PMID:18166083

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

PubMed Central

Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

2013-01-01

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

PubMed Central

Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

1982-01-01

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

PubMed Central

Kishor, Aparna; Tandukar, Bishal; Ly, Yann V.; Toth, Eric A.; Suarez, Yvelisse; Brewer, Gary

2013-01-01

The AU-rich elements (AREs) encoded within many mRNA 3′ untranslated regions (3′UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3′UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes. PMID:23109422

A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells

PubMed Central

Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S.

2015-01-01

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. PMID:25845589

Sex Differences in Behavior and Expression of CGRP-related Genes in a Rodent Model of Chronic Migraine

PubMed Central

Stucky, Nicholas L.; Gregory, Eugene; Winter, Michelle K.; He, Yong-Yue; Hamilton, Eric S.; McCarson, Kenneth E.; Berman, Nancy E.J.

2014-01-01

a. Objective The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of CGRP and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. b. Background Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (PBS) to the dura were measured to model changes in routine activity and allodynia. Calcitonin gene related peptide (CGRP), an important mediator of migraine pathogenesis, and the three components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. c. Methods Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 microliter volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 min post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. d. Results Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µl IS groups), and males showed increased perimasseter withdrawal responses (20 µl IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP related genes were upregulated by both IS and PBS applications. e. Conclusions This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation. PMID:21521205

Identification and characterization of long non-coding RNAs in rainbow trout eggs

USDA-ARS?s Scientific Manuscript database

Long non-coding RNAs (lncRNAs) are in general considered as a diverse class of transcripts longer than 200 nucleotides that structurally resemble mRNAs but do not encode proteins. Recent advances in RNA sequencing (RNA-Seq) and bioinformatics methods have provided an opportunity to indentify and ana...

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

PubMed

Teng, Chang Ying; Dang, Yunkun; Danne, Jillian C; Waller, Ross F; Green, Beverley R

2013-01-01

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

The DEAD-box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish.

PubMed

Zhang, Linlin; Yang, Yuxi; Li, Beibei; Scott, Ian C; Lou, Xin

2018-04-23

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism, including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease remain largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a mutant that disrupts the gene encoding the RNA helicase DEAD-box 39ab ( ddx39ab ). Homozygous ddx39ab mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities, caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Loss of ddx39ab hindered splicing of mRNAs encoding epigenetic regulatory factors, including members of the KMT2 gene family, leading to misregulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39ab plays an essential role in establishment of the proper epigenetic status during differentiation of multiple cell lineages. © 2018. Published by The Company of Biologists Ltd.

Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins.

PubMed

Makeyev, A V; Liebhaber, S A

2000-08-01

We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, alphaCP-1 and alphaCP-2. Both of these novel genes, alphaCP-3 and alphaCP-4, are predicted to encode proteins with triplicated KH domains. The number and organization of the KH domains, their sequences, and the sequences of the contiguous regions are conserved among all four alphaCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon-intron organization in the corresponding genes. The map positions of alphaCP-1 and alphaCP-2 (previously reported) and those of alphaCP-3 and alphaCP-4 (present report) reveal that the four alphaCP loci are dispersed in the human genome; alphaCP-3 and alphaCP-4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as alphaCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21-q22). Although the overall levels of alphaCP-3 and alphaCP-4 mRNAs are substantially lower than those of alphaCP-1 and alphaCP-2, transcripts of alphaCP-3 and alphaCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls. Copyright 2000 Academic Press.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

PubMed

Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

2016-04-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

PubMed Central

Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

2016-01-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

PubMed

Kulczykowska, Ewa; Kleszczyńska, Agnieszka; Gozdowska, Magdalena; Sokołowska, Ewa

2017-06-01

In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS.

PubMed

Blumhagen, Rachel Z; Hedin, Brenna R; Malcolm, Kenneth C; Burnham, Ellen L; Moss, Marc; Abraham, Edward; Huie, Tristan J; Nick, Jerry A; Fingerlin, Tasha E; Alper, Scott

2017-11-01

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1 , two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88 L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88 S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches. Copyright © 2017 the American Physiological Society.

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): II. Fetal rat brain.

PubMed

Pimenta, A F; Reinoso, B S; Levitt, P

1996-11-11

The limbic system-associated membrane protein (LAMP) is a 64-68 kDa neuronal surface glycoprotein expressed in cortical and subcortical regions of the limbic system of the adult and developing rat central nervous system (CNS). LAMP is a member of the immunoglobulin superfamily of cell adhesion molecules with three Ig domains and is highly conserved between rat and human. In this study, the temporal and spatial pattern of lamp gene expression during fetal rat development was analyzed by using Northern blot analysis and in situ hybridization. In Northern blot analysis, two lamp mRNA transcripts, 1.6 kb and 8.0 kb, identical in size to those present in the adult rat nervous system, were detected in developing neural tissue. In situ hybridization analysis showed close correlation, though not identity, between the expression of lamp mRNAs and the distribution of LAMP in limbic regions of the developing rat CNS, indicative of a more complex regulation of gene expression than was previously thought to be the case. The expression of lamp mRNAs is first detected on about embryonic day (E) 13. The hybridization signal is not seen in the proliferative ventricular zone at any level of the neuraxis, indicating that lamp is expressed in postmitotic neurons. In the cerebral cortex, lamp mRNAs are expressed in limbic cortical regions, such as the perirhinal cortex, prefrontal cortex, and cingulate cortex. In the hippocampus, the hybridization signal is observed in Ammon's horn by E18. The neostriatum, amygdaloid complex, and most hypothalamic areas express lamp mRNAs from early stages (E13-E14) in a pattern consistent with the onset of neurogenesis. The emerging patterns of lamp expression at the outset are similar to those seen in adult hypothalamus and dorsal thalamus. Although the hybridization signal is observed in some nonlimbic areas, including midbrain and hindbrain structures, intense labeling is evident in more classic limbic regions. The high levels of expression of lamp in limbic regions, beginning in early developmental stages, combined with the results of previous functional in vitro and in vivo studies, support a role for LAMP as a recognition molecule involved in the formation of limbic connections.

Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans

PubMed Central

Morrison, J Kaitlin; Friday, Andrew J; Henderson, Melissa A; Hao, Enhui; Keiper, Brett D

2014-01-01

During apoptosis, activated caspases cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation within the cell. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. Many of these mRNAs, including cell death related mRNAs, contain internal ribosome entry sites (IRESes) that promote their enhanced translation during apoptosis. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, allows for an initial period of attempted cell survival, then commitment to cell death when damage is extensive. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis. PMID:26779406

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.

PubMed

Vallery, Tenaya K; Withers, Johanna B; Andoh, Joana A; Steitz, Joan A

2018-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments. IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus. Copyright © 2018 American Society for Microbiology.

Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition.

PubMed

Laver, John D; Li, Xiao; Ray, Debashish; Cook, Kate B; Hahn, Noah A; Nabeel-Shah, Syed; Kekis, Mariana; Luo, Hua; Marsolais, Alexander J; Fung, Karen Yy; Hughes, Timothy R; Westwood, J Timothy; Sidhu, Sachdev S; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2015-05-12

Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Expression of CXCL4 and aquaporin 3 and 10 mRNAs in patients with otitis media with effusion.

PubMed

Jin, Zhe; Cha, Sung Ho; Choi, Yong-Sung; Kim, Young Il; Choi, Sun A; Yeo, Seung Geun

2016-02-01

Bacterial infections in children with underdeveloped Eustachian tubes are a major cause of otitis media with effusion (OEM), and persistent effusion in the middle ear in these patients is a major cause of surgical intervention. CXCL4 is associated with bacterial infection, and aquaporins 3 and 10 are associated with water metabolism. This study assessed the expression of mRNAs encoding CXCL-4 and aquaporins 3 and 10 in the effusion of pediatric OME patients, and the association of this expression with clinical manifestations. Levels of CXCL4 and aquaporin 3 and 10 mRNA were assayed by real-time RT-PCR in the middle ear effusion of 38 pediatric patients with OME requiring ventilation tube insertion. The relationships of these mRNA levels with the presence of bacteria; concomitant diseases such as allergic rhinitis, sinusitis, and adenoid disease; recurrence of OME; and number of ventilation tube insertions were evaluated. CXCL4 and aquaporin 3 and 10 mRNAs were expressed in middle ear effusion of all OME patients. CXCL-4 mRNA levels were significantly lower when bacteria were present and in patients with concomitant diseases (p<0.05 each). Levels of all three mRNAs were unrelated to OME recurrence or number of ventilation tube insertions (p>0.05 each). The levels of CXCL4 and aquaporin 10 mRNAs were significantly correlated (p<0.05). Expression of CXCL4 and aquaporin 3 and 10 mRNAs in middle ear effusion is associated with the pathophysiology of OME. CXCL4 mRNA levels are significantly lower in patients with than without concomitant diseases or bacterial infections. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

5’-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation

PubMed Central

Beck, Heather J.; Fleming, Ian M. C.

2016-01-01

Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene’s start codon while also containing an AUG triplet at the mRNA’s 5’- terminus (5’-uAUG). Fusion of the coding sequence specified by the 5’-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5’-terminal upstream open reading frames (5’-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5’-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5’-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5’-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5’-uORFs may play roles in downstream regulation. Since the 5’-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5’-uAUGs should be examined for their potential to function also as leaderless mRNAs. PMID:27467758

Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system

PubMed Central

Ross, Joseph A.; Ellis, Michael J.; Hossain, Shahan; Haniford, David B.

2013-01-01

Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements. PMID:23510801

Molecular cloning and characterization of a new basic peroxidase cDNA from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines.

PubMed

Yi, S Y; Hwang, B K

1998-10-31

Differential display techniques were used to isolate cDNA clones corresponding to genes which were expressed in soybean hypocotyls by Phytophthora sojae f.sp. glycines infection. With a partial cDNA clone C20CI4 from the differential display PCR as a probe, a new basic peroxidase cDNA clone, designated GMIPER1, was isolated from a cDNA library of soybean hypocotyls infected with P. sojae f.sp. glycines. Sequence analysis revealed that the peroxidase clone encodes a mature protein of 35,813 Da with a putative signal peptide of 27 amino acids in its N-terminus. The amino acid sequence of the soybean peroxidase GMIPER1 is between 54-75% identical to other plant peroxidases including a soybean seed coat peroxidase. Southern blot analysis indicated that multiple copies of sequences related to GMIPER1 exist in the soybean genome. The mRNAs corresponding to the GMIPER1 cDNA accumulated predominantly in the soybean hypocotyls infected with the incompatible race of P. sojae f.sp. glycines, but were expressed at low levels in the compatible interaction. Soybean GMIPER1 mRNAs were not expressed in hypocotyls, leaves, stems, and roots of soybean seedlings. However, treatments with ethephon, salicylic acid or methyl jasmonate induced the accumulation of the GMIPER1 mRNAs in the different organs of soybean. These results suggest that the GMIPER1 gene encoding a putative pathogen-induced peroxidase may play an important role in induced resistance of soybean to P. sojae f.sp. glycines and in response to various external stresses.

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

PubMed

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

Transient Early Embryonic Expression of Nkx2-5 Mutations Linked to Congenital Heart Defects in Human Causes Heart Defects in Xenopus laevis

PubMed Central

Bartlett, Heather L.; Sutherland, Lillian; Kolker, Sandra J.; Welp, Chelsea; Tajchman, Urszula; Desmarais, Vera; Weeks, Daniel L.

2007-01-01

Nkx2-5 is a homeobox containing transcription factor that is conserved and expressed in organisms that form hearts. Fruit flies lacking the gene (tinman) fail to form a dorsal vessel, mice that are homozygous null for Nkx2-5 form small, deformed hearts, and several human cardiac defects have been linked to dominant mutations in the Nkx2-5 gene. The Xenopus homologs (XNkx2-5) of two truncated forms of Nkx2-5 that have been identified in humans with congenital heart defects were used in the studies reported here. mRNAs encoding these mutations were injected into single cell Xenopus embryos, and heart development was monitored. Our results indicate that the introduction of truncated XNkx2-5 variants leads to three principle developmental defects. The atrial septum and the valve of the atrioventricular canal were both abnormal. In addition, video microscopic timing of heart contraction indicated that embryos injected with either mutant form of XNkx2-5 have conduction defects. PMID:17685485

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

PubMed Central

Rezelj, Veronica V.; Elliott, Richard M.

2017-01-01

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA. PMID:28592543

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses.

PubMed

Brennan, Benjamin; Rezelj, Veronica V; Elliott, Richard M

2017-08-15

SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3' rapid amplification of cDNA ends (RACE), we mapped the 3' end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3' end of the N mRNA terminates upstream of a 5'-GCCAGCC-3' motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5'-GCCAGCC-3' motif present in the virus genomic S RNA. Copyright © 2017 Brennan et al.

Changes of lysosomes in the earliest stages of the development of atherosclerosis.

PubMed

Bobryshev, Yuri V; Shchelkunova, Tatyana A; Morozov, Ivan A; Rubtsov, Petr M; Sobenin, Igor A; Orekhov, Alexander N; Smirnov, Alexander N

2013-05-01

One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the 'normal intima-initial lesion-fatty streak' sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease. © 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

PubMed Central

Duronio, Robert J.; Marzluff, William F.

2017-01-01

ABSTRACT Metazoan replication-dependent (RD) histone genes encode the only known cellular mRNAs that are not polyadenylated. These mRNAs end instead in a conserved stem-loop, which is formed by an endonucleolytic cleavage of the pre-mRNA. The genes for all 5 histone proteins are clustered in all metazoans and coordinately regulated with high levels of expression during S phase. Production of histone mRNAs occurs in a nuclear body called the Histone Locus Body (HLB), a subdomain of the nucleus defined by a concentration of factors necessary for histone gene transcription and pre-mRNA processing. These factors include the scaffolding protein NPAT, essential for histone gene transcription, and FLASH and U7 snRNP, both essential for histone pre-mRNA processing. Histone gene expression is activated by Cyclin E/Cdk2-mediated phosphorylation of NPAT at the G1-S transition. The concentration of factors within the HLB couples transcription with pre-mRNA processing, enhancing the efficiency of histone mRNA biosynthesis. PMID:28059623

A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

PubMed

Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

2015-04-30

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Aβ1-42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons.

PubMed

Walker, Chandler A; Randolph, Lisa K; Matute, Carlos; Alberdi, Elena; Baleriola, Jimena; Hengst, Ulrich

2018-05-14

Neurons frequently encounter neurodegenerative signals first in their periphery. For example, exposure of axons to oligomeric Aβ 1-42 is sufficient to induce changes in the neuronal cell body that ultimately lead to degeneration. Currently, it is unclear how the information about the neurodegenerative insult is transmitted to the soma. Here, we find that the translation of pre-localized but normally silenced sentinel mRNAs in axons is induced within minutes of Aβ 1-42 addition in a Ca 2+ -dependent manner. This immediate protein synthesis following Aβ 1-42 exposure generates a retrograde signaling complex including vimentin. Inhibition of the immediate protein synthesis, knock-down of axonal vimentin synthesis, or inhibition of dynein-dependent transport to the soma prevented the normal cell body response to Aβ 1-42 These results establish that CNS axons react to neurodegenerative insults via the local translation of sentinel mRNAs encoding components of a retrograde signaling complex that transmit the information about the event to the neuronal soma. © 2018 The Authors.

Matrix Metalloproteinase Dysregulation in the Stria Vascularis of Mice with Alport Syndrome

PubMed Central

Gratton, Michael Anne; Rao, Velidi H.; Meehan, Daniel T.; Askew, Charles; Cosgrove, Dominic

2005-01-01

Alport syndrome results from mutations in genes encoding collagen α3(IV), α4(IV), or α5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism. PMID:15855646

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

PubMed Central

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V.

2012-01-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3′-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PMID:22661231

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

PubMed

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V

2012-06-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Molecular control of vertebrate iron homeostasis by iron regulatory proteins

PubMed Central

Wallander, Michelle L.; Leibold, Elizabeth A.; Eisenstein, Richard S.

2008-01-01

Both deficiencies and excesses of iron represent major public health problems throughout the world. Understanding the cellular and organismal processes controlling iron homeostasis is critical for identifying iron-related diseases and in advancing the clinical treatments for such disorders of iron metabolism. Iron regulatory proteins (IRPs) 1 and 2 are key regulators of vertebrate iron metabolism. These RNA binding proteins post-transcriptionally control the stability or translation of mRNAs encoding proteins involved in iron homeostasis thereby controlling the uptake, utilization, storage or export of iron. Recent evidence provides insight into how IRPs selectively control the translation or stability of target mRNAs, how IRP RNA binding activity is controlled by iron-dependent and iron-independent effectors, and the pathological consequences of dysregulation of the IRP system. PMID:16872694

Epitranscriptional orchestration of genetic reprogramming is an emergent property of stress-regulated cardiac microRNAs

PubMed Central

Hu, Yuanxin; Matkovich, Scot J.; Hecker, Peter A.; Zhang, Yan; Edwards, John R.; Dorn, Gerald W.

2012-01-01

Cardiac stress responses are driven by an evolutionarily conserved gene expression program comprising dozens of microRNAs and hundreds of mRNAs. Functionalities of different individual microRNAs are being studied, but the overall purpose of interactions between stress-regulated microRNAs and mRNAs and potentially distinct roles for microRNA-mediated epigenetic and conventional transcriptional genetic reprogramming of the stressed heart are unknown. Here we used deep sequencing to interrogate microRNA and mRNA regulation in pressure-overloaded mouse hearts, and performed a genome-wide examination of microRNA–mRNA interactions during early cardiac hypertrophy. Based on abundance and regulatory patterns, cardiac microRNAs were categorized as constitutively expressed housekeeping, regulated homeostatic, or dynamic early stress-responsive microRNAs. Regulation of 62 stress-responsive cardiac microRNAs directly affected levels of only 66 mRNAs, but the global impact of microRNA-mediated epigenetic regulation was amplified by preferential targeting of mRNAs encoding transcription factors, kinases, and phosphatases exerting amplified secondary effects. Thus, an emergent cooperative property of stress-regulated microRNAs is orchestration of transcriptional and posttranslational events that help determine the stress-reactive cardiac phenotype. This global functionality explains how large end-organ effects can be induced through modest individual changes in target mRNA and protein content by microRNAs that sense and respond dynamically to a changing physiological milieu. PMID:23150554

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs.

PubMed

Shahid, Saima; Kim, Gunjune; Johnson, Nathan R; Wafula, Eric; Wang, Feng; Coruh, Ceyda; Bernal-Galeano, Vivian; Phifer, Tamia; dePamphilis, Claude W; Westwood, James H; Axtell, Michael J

2018-01-03

Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite, but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana. Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris. The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana. Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis.

PubMed

Karamysheva, Zemfira N; Tikhonova, Elena B; Grozdanov, Petar N; Huffman, James C; Baca, Kristen R; Karamyshev, Alexander; Denison, R Brian; MacDonald, Clinton C; Zhang, Kai; Karamyshev, Andrey L

2018-04-08

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

PubMed

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells

PubMed Central

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A.; Twiss, Jeffery L.

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5’ untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5’ UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality. PMID:27224031

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

PubMed

Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

2017-09-19

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs

PubMed Central

Kim, Eugene Z.; Wespiser, Adam R.; Caffrey, Daniel R.

2016-01-01

Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3′ and 5′ ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions. PMID:26654912

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

PubMed Central

2012-01-01

Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

PubMed Central

Soreq, H; Zevin-Sonkin, D; Razon, N

1984-01-01

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA. PMID:6745236

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

PubMed

Krebber, H; Taura, T; Lee, M S; Silver, P A

1999-08-01

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

The Unexpected Tuners: Are LncRNAs Regulating Host Translation during Infections?

PubMed Central

Knap, Primoz; Tebaldi, Toma; Di Leva, Francesca; Biagioli, Marta; Dalla Serra, Mauro; Viero, Gabriella

2017-01-01

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells—i.e., host translation—to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections. PMID:29469820

New Method for Producing Significant Amounts of RNA Labeled at Specific Sites | Center for Cancer Research

Cancer.gov

Among biomacromolecules, RNA is the most versatile, and it plays indispensable roles in almost all aspects of biology. For example, in addition to serving as mRNAs coding for proteins, RNAs regulate gene expression, such as controlling where, when, and how efficiently a gene gets expressed, participate in RNA processing, encode the genetic information of some viruses, serve as

DUSP1 and KCNJ2 mRNA upregulation can serve as a biomarker of mechanical asphyxia-induced death in cardiac tissue.

PubMed

Zeng, Yan; Tao, Li; Ma, Jianlong; Han, Liujun; Lv, Yehui; Hui, Pan; Zhang, Heng; Ma, Kaijun; Xiao, Bi; Shi, Qun; Xu, Hongmei; Chen, Long

2018-05-01

The incidence of death by asphyxia is second to the incidence of death by mechanical injury; however, death by mechanical asphyxia may be difficult to prove in court, particularly in cases in which corpses do not exhibit obvious signs of asphyxia. To identify a credible biomarker of asphyxia, we first examined the expression levels of 47,000 mRNAs in human cardiac tissue specimens from individuals who died of mechanical asphyxia and compared the expression levels with the levels of the corresponding mRNAs in specimens from individuals who died of craniocerebral injury using microarray. We selected 119 differentially expressed mRNAs, examined the expression levels of these mRNAs in 44 human cardiac tissue specimens of individuals who died of mechanical asphyxia, craniocerebral injury, hemorrhagic shock, or other causes. That the expression of dual-specificity phosphatase 1 (DUSP1) and potassium voltage-gated channel subfamily J member 2 (KCNJ2) was upregulated in human cardiac tissues from the mechanical asphyxia group compared with control tissues, regardless of age, environmental temperature, and postmortem interval (PMI), indicating that DUSP1 and KCNJ2 may be associated with mechanical asphyxia-induced death and can thus serve as useful biomarkers of death by mechanical asphyxia.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

PubMed Central

Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

2013-01-01

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. PMID:24345856

Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

PubMed Central

Popp, Maximilian Wei-Lin; Maquat, Lynne E.

2014-01-01

Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

Final Scientific/Technical Report for DOE Award No. DE-FG02-03ER15426: Role of Arabidopsis PINHEAD gene in meristem function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dr. M. Kathryn Barton

2011-11-29

The shoot apical meristems of land plants are small mounds of hundreds of cells located at the tips of branches. It is from these small clusters of cells that essentially all above ground plant biomass and therefore much of our energy supply originates. Several key genes have been discovered that are necessary for cells in the shoot apical meristem to take on stem cell properties. The goal of this project is to understand how the synthesis and accumulation of the mRNAs and proteins encoded by these genes is controlled. A thorough understanding of the molecules that control the growth ofmore » shoot apical meristems in plants will help us to manipulate food, fiber and biofuel crops to better feed, clothe and provide energy for humans.« less

Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes.

PubMed

Colson, Philippe; La Scola, Bernard; Raoult, Didier

2017-09-29

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

PubMed Central

Biever, Anne; Boubaker-Vitre, Jihane; Cutando, Laura; Gracia-Rubio, Irene; Costa-Mattioli, Mauro; Puighermanal, Emma; Valjent, Emmanuel

2017-01-01

Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum. PMID:28119566

Differential Accumulation of Sunflower Tetraubiquitin mRNAs during Zygotic Embryogenesis and Developmental Regulation of Their Heat-Shock Response.

PubMed Central

Almoguera, C.; Coca, M. A.; Jordano, J.

1995-01-01

We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed. PMID:12228401

Cell-Type-Specific Translation Profiling Reveals a Novel Strategy for Treating Fragile X Syndrome.

PubMed

Thomson, Sophie R; Seo, Sang S; Barnes, Stephanie A; Louros, Susana R; Muscas, Melania; Dando, Owen; Kirby, Caoimhe; Wyllie, David J A; Hardingham, Giles E; Kind, Peter C; Osterweil, Emily K

2017-08-02

Excessive mRNA translation downstream of group I metabotropic glutamate receptors (mGlu 1/5 ) is a core pathophysiology of fragile X syndrome (FX); however, the differentially translating mRNAs that contribute to altered neural function are not known. We used translating ribosome affinity purification (TRAP) and RNA-seq to identify mistranslating mRNAs in CA1 pyramidal neurons of the FX mouse model (Fmr1 -/y ) hippocampus, which exhibit exaggerated mGlu 1/5 -induced long-term synaptic depression (LTD). In these neurons, we find that the Chrm4 transcript encoding muscarinic acetylcholine receptor 4 (M 4 ) is excessively translated, and synthesis of M 4 downstream of mGlu 5 activation is mimicked and occluded. Surprisingly, enhancement rather than inhibition of M 4 activity normalizes core phenotypes in the Fmr1 -/y , including excessive protein synthesis, exaggerated mGluR-LTD, and audiogenic seizures. These results suggest that not all excessively translated mRNAs in the Fmr1 -/y brain are detrimental, and some may be candidates for enhancement to correct pathological changes in the FX brain. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

PubMed Central

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-01-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.

PubMed

Fritz, Melanie; Vanselow, Jens; Sauer, Nadja; Lamer, Stephanie; Goos, Carina; Siegel, T Nicolai; Subota, Ines; Schlosser, Andreas; Carrington, Mark; Kramer, Susanne

2015-09-18

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Minute virus of mice (MVM) mRNAs predominantly polyadenylate at a single site.

PubMed

Clemens, K E; Pintel, D

1987-10-01

The polyadenylation sites for MVM(p) and MVM(i) mRNAs were determined by a quantitative hybridization-S1 protection assay. mRNAs produced by MVM(p) both early and late in infection of mouse A9 fibroblasts, and by MVM(p) and MVM(i) late in infection of human NB324K cells, polyadenylate predominantly at a single site, at nucleotide 4908 +/- 2 for MVM(p) and 4843 +/- 2 for MVM(i), shortly downstream of the final AATAAA in each viral genome. These results demonstrate that although the right-hand end of MVM has multiple AATAAA signals, and MVM(p) and MVM(i) vary significantly within this region, 3' end processing of viral mRNAs is not a prevalent mechanism for the regulation of MVM gene expression.

Maternally Recruited DCP1A and DCP2 Contribute to Messenger RNA Degradation During Oocyte Maturation and Genome Activation in Mouse1

PubMed Central

Ma, Jun; Flemr, Matyas; Strnad, Hynek; Svoboda, Petr; Schultz, Richard M.

2012-01-01

ABSTRACT The oocyte-to-zygote transition entails transforming a highly differentiated oocyte into totipotent blastomeres and represents one of the earliest obstacles that must be successfully hurdled for continued development. Degradation of maternal mRNAs, which likely lies at the heart of this transition, is characterized by a transition from mRNA stability to instability during oocyte maturation. Although phosphorylation of the oocyte-specific RNA-binding protein MSY2 during maturation is implicated in making maternal mRNAs more susceptible to degradation, mechanisms underlying mRNA degradation during oocyte maturation remain poorly understood. We report that DCP1A and DCP2, proteins responsible for decapping mRNA, are encoded by maternal mRNAs recruited for translation during maturation via cytoplasmic polyadenylation elements located in their 3′ untranslated regions. Both DCP1A and DCP2 are phosphorylated during maturation, with CDC2A being the kinase likely responsible for both, although MAPK may be involved in DCP1A phosphorylation. Inhibiting accumulation of DCP1A and DCP2 by RNA interference or morpholinos decreases not only degradation of mRNAs during meiotic maturation but also transcription of the zygotic genome. The results indicate that maternally recruited DCP1A and DCP2 are critical players in the transition from mRNA stability to instability during meiotic maturation and that proper maternal mRNA degradation must be successful to execute the oocyte-to-zygote transition. PMID:23136299

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

NASA Technical Reports Server (NTRS)

Shibata, K.; Abe, S.; Davies, E.

2001-01-01

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain.

PubMed

Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D

2008-12-01

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

Advances in RNA Structure Determination | Center for Cancer Research

Cancer.gov

The recent years have witnessed a revolution in the field of RNA structure and function. Until recently the main contribution of RNA in cellular and disease functions was considered to be a role defined by the central dogma, namely DNA codes for mRNAs, which in turn encode for proteins, a notion facilitated by non-coding ribosomal RNA and tRNA. It was also assumed at the time

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability*

PubMed Central

Howard, Michael T.; Carlson, Bradley A.; Anderson, Christine B.; Hatfield, Dolph L.

2013-01-01

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. PMID:23696641

Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation.

PubMed

Hood, D A; Simoneau, J A; Kelly, A M; Pette, D

1992-10-01

The effect of thyroid status on the expression of cytochrome c oxidase (CYTOX) and the activities of citrate synthase (CS) and phosphofructokinase (PFK) were examined in chronically stimulated (10 Hz; 35 days) and contralateral, nonstimulated rat tibialis anterior muscle of hypothyroid, hyperthyroid, and euthyroid animals. Stimulation increased CYTOX activity by 2.7-, 3.2-, and 4.9-fold in hyperthyroid, euthyroid, and hypothyroid animals, respectively, to similar absolute values. CS displayed similar increases. Stimulation reduced PFK activity in hypothyroid and euthyroid animals to 45% and 60% of control values. This effect was abolished with hyperthyroidism. Thus stimulation and thyroid hormone act antagonistically on PFK activity. Stimulation increased CYTOX subunit III (mitochondrially encoded) mRNA by 2.5- and 2.9-fold in hyperthyroid and euthyroid animals. Similar increases were observed in the nuclear-encoded mRNAs of CYTOX subunit VIc in euthyroid muscle. In hyperthyroid and euthyroid conditions, the mRNA changes paralleled the increases in enzyme activity. In hypothyroid muscle, the increase in mRNA was less for subunit VIc than III, suggesting that hypothyroidism upsets the coordinate expression of nuclear and mitochondrial genes. Further, the increases in CYTOX activity exceeded that of both subunit mRNAs in hypothyroid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

A trans-acting leader RNA from a Salmonella virulence gene

PubMed Central

Choi, Eunna; Han, Yoontak; Cho, Yong-Joon; Nam, Daesil; Lee, Eun-Jin

2017-01-01

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection. PMID:28874555

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

PubMed Central

Chen, E. Ricky; Nilsen, Timothy W.

2017-01-01

ABSTRACT N6-adenosine methylation (m6A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m6A modification. The levels of m6A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m6A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m6A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m6A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication. PMID:28592530

The translational regulator Cup controls NMJ presynaptic terminal morphology.

PubMed

Menon, Kaushiki P; Carrillo, Robert A; Zinn, Kai

2015-07-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. Copyright © 2015 Elsevier Inc. All rights reserved.

The translational regulator Cup controls NMJ presynaptic terminal morphology

PubMed Central

Menon, Kaushiki P.; Carrillo, Robert A.; Zinn, Kai

2015-01-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with four genes (EndoA, WASp, Dap160, and Synj) encoding proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. PMID:26102195

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes

PubMed Central

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-01-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau155-HA, Stau259-HA, or Stau262-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau259-HA- and Stau262-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptionnal gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs. PMID:18094122

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes.

PubMed

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-02-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau1(55)-HA, Stau2(59)-HA, or Stau2(62)-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau2(59)-HA- and Stau2(62)-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptional gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs.

Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3).

PubMed

Race, J E; Grassl, S M; Williams, W J; Holtzman, E J

1999-02-16

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide. Copyright 1999 Academic Press.

Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes

PubMed Central

Eberle, Andrea B.; Hessle, Viktoria; Helbig, Roger; Dantoft, Widad; Gimber, Niclas; Visa, Neus

2010-01-01

Background Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. Methodology/Principal Findings We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-globin transcripts are much lower than those of wild type (wt) ß-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut β-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut β-globin gene shows reduced levels of H3K4me3. Conclusions/Significance Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. PMID:20634951

3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation

PubMed Central

Fitzpatrick, Terry; Huang, Sui

2012-01-01

Alu repeats within human genes may potentially alter gene expression. Here, we show that 3′-UTR-located inverted Alu repeats significantly reduce expression of an AcGFP reporter gene. Mutational analysis demonstrates that the secondary structure, but not the primary nucleotide sequence, of the inverted Alu repeats is critical for repression. The expression levels and nucleocytoplasmic distribution of reporter mRNAs with or without 3′-UTR inverted Alu repeats are similar; suggesting that reporter gene repression is not due to changes in mRNA levels or mRNA nuclear sequestration. Instead, reporter gene mRNAs harboring 3′-UTR inverted Alu repeats accumulate in cytoplasmic stress granules. These findings may suggest a novel mechanism whereby 3′-UTR-located inverted Alu repeats regulate human gene expression through sequestration of mRNAs within stress granules. PMID:22688648

Expression Profile of Long Noncoding RNAs in Human Earlobe Keloids: A Microarray Analysis

PubMed Central

Guo, Liang; Xu, Kai; Yan, Hongbo; Feng, Haifeng

2016-01-01

Background. Long noncoding RNAs (lncRNAs) play key roles in a wide range of biological processes and their deregulation results in human disease, including keloids. Earlobe keloid is a type of pathological skin scar, and the molecular pathogenesis of this disease remains largely unknown. Methods. In this study, microarray analysis was used to determine the expression profiles of lncRNAs and mRNAs between 3 pairs of earlobe keloid and normal specimens. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the main functions of the differentially expressed genes and earlobe keloid-related pathways. Results. A total of 2068 lncRNAs and 1511 mRNAs were differentially expressed between earlobe keloid and normal tissues. Among them, 1290 lncRNAs and 1092 mRNAs were upregulated, and 778 lncRNAs and 419 mRNAs were downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated transcripts, while 11 pathways were associated with the downregulated transcripts. Conclusion. We characterized the expression profiles of lncRNA and mRNA in earlobe keloids and suggest that lncRNAs may serve as diagnostic biomarkers for the therapy of earlobe keloid. PMID:28101509

Hacking RNA: Hakai promotes tumorigenesis by switching on the RNA-binding function of PSF

PubMed Central

Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

2009-01-01

Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF’s RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF’s ability to bind RNAs that promote cancer-related gene expression. PMID:19855157

Function of alternative splicing

PubMed Central

Kelemen, Olga; Convertini, Paolo; Zhang, Zhaiyi; Wen, Yuan; Shen, Manli; Falaleeva, Marina; Stamm, Stefan

2017-01-01

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed. PMID:22909801

A mRNA and cognate microRNAs localize in the nucleolus.

PubMed

Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Sequence Divergence in the 3′ Untranslated Regions of Human ζ- and α-Globin mRNAs Mediates a Difference in Their Stabilities and Contributes to Efficient α-to-ζ Gene Developmental Switching

PubMed Central

Russell, J. Eric; Morales, Julia; Makeyev, Aleksandr V.; Liebhaber, Stephen A.

1998-01-01

The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of ζ- and α-globin in the embryonic yolk sac to exclusive expression of α-globin during fetal and adult life. Recent studies with transgenic mice demonstrate that in addition to transcriptional control elements, full developmental silencing of the human ζ-globin gene requires elements encoded within the transcribed region. In the current work, we establish that these latter elements operate posttranscriptionally by reducing the relative stability of ζ-globin mRNA. Using a transgenic mouse model system, we demonstrate that human ζ-globin mRNA is unstable in adult erythroid cells relative to the highly stable human α-globin mRNA. A critical determinant of the difference between α- and ζ-globin mRNA stability is mapped by in vivo expression studies to their respective 3′ untranslated regions (3′UTRs). In vitro messenger ribonucleoprotein (mRNP) assembly assays demonstrate that the α- and ζ-globin 3′UTRs assemble a previously described mRNP stability-determining complex, the α-complex, with distinctly different affinities. The diminished efficiency of α-complex assembly on the ζ 3′UTR results from a single C→G nucleotide substitution in a crucial polypyrimidine tract contained by both the human α- and ζ-globin mRNA 3′UTRs. A potential pathway for accelerated ζ-globin mRNA decay is suggested by the observation that its 3′UTR encodes a shortened poly(A) tail. Based upon these data, we propose a model for ζ-globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of ζ-globin transcripts. PMID:9528789

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

Tissue-specific thyroid hormone regulation of gene transcripts encoding iodothyronine deiodinases and thyroid hormone receptors in striped parrotfish (Scarus iseri).

PubMed

Johnson, Kaitlin M; Lema, Sean C

2011-07-01

In fish as in other vertebrates, the diverse functions of thyroid hormones are mediated at the peripheral tissue level through iodothyronine deiodinase (dio) enzymes and thyroid hormone receptor (tr) proteins. In this study, we examined thyroid hormone regulation of mRNAs encoding the three deiodinases dio1, dio2 and dio3 - as well as three thyroid hormone receptors trαA, trαB and trβ - in initial phase striped parrotfish (Scarus iseri). Parrotfish were treated with dissolved phase T(3) (20 nM) or methimazole (3 mM) for 3 days. Treatment with exogenous T(3) elevated circulating T(3), while the methimazole treatment depressed plasma T(4). Experimentally-induced hyperthyroidism increased the relative abundance of transcripts encoding trαA and trβ in the liver and brain, but did not affect trαB mRNA levels in either tissue. In both sexes, methimazole-treated fish exhibited elevated dio2 transcripts in the liver and brain, suggesting enhanced outer-ring deiodination activity in these tissues. Accordingly, systemic hyperthyroidism elevated relative dio3 transcript levels in these same tissues. In the gonad, however, patterns of transcript regulation were distinctly different with elevated T(3) increasing mRNAs encoding dio2 in testicular and ovarian tissues and dio3, trαA and trαB in the testes only. Thyroid hormone status did not affect dio1 transcript abundance in the liver, brain or gonads. Taken as a whole, these results demonstrate that thyroidal status influences relative transcript abundance for dio2 and dio3 in the liver, provide new evidence for similar patterns of dio2 and dio3 mRNA regulation in the brain, and make evident that fish exhibit tr subtype-specific transcript abundance changes to altered thyroid status. Copyright © 2011 Elsevier Inc. All rights reserved.

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

PubMed Central

Liu, X; Gorovsky, M A

1996-01-01

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

PubMed

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Upf1 senses 3′UTR length to potentiate mRNA decay

PubMed Central

Hogg, J. Robert; Goff, Stephen P.

2010-01-01

Summary The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. PMID:21029861

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

PubMed

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-10-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae

PubMed Central

Davis, Carrie Anne; Ares, Manuel

2006-01-01

Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Δ yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of ≈250–500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional “noise,” we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus. PMID:16484372

Inflammation in Lafora Disease: Evolution with Disease Progression in Laforin and Malin Knock-out Mouse Models.

PubMed

López-González, Irene; Viana, Rosa; Sanz, Pascual; Ferrer, Isidre

2017-07-01

Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a -/- (laforin knock-out) and Epm2b -/- (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 messenger RNAs (mRNAs) are significantly increased in Epm2a -/- mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα, and Il10ra mRNAs are significantly upregulated in Epm2b -/- at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα, and Cox2 particularly in Epm2b -/- mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b -/- mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD.

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

PubMed

Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

2014-01-01

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σ(E2) or σ(H1/2). Recovery rates of sRNAs in each of the CoIP-RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σ(E2)-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5' regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA-mRNA regulatory pairs.

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti

PubMed Central

Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

2014-01-01

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σE2 or σH1/2. Recovery rates of sRNAs in each of the CoIP–RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σE2-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5′ regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA–mRNA regulatory pairs. PMID:24786641

Changes in the levels of mRNAs for GH/prolactin/somatolactin family and Pit-1/GHF-1 in the pituitaries of pre-spawning chum salmon.

PubMed

Taniyama, S; Kitahashi, T; Ando, H; Ban, M; Ueda, H; Urano, A

1999-10-01

Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration.

RRP42, a Subunit of Exosome, Plays an Important Role in Female Gametophytes Development and Mesophyll Cell Morphogenesis in Arabidopsis.

PubMed

Yan, Xiaoyuan; Yan, Zongyun; Han, Yuzhen

2017-01-01

The exosome complex plays a central and essential role in RNA metabolism. However, current research on functions of exosome subunit in plants is limited. Here, we used an egg cell-specific promoter-controlled CRISPR/Cas9 system to knock out RRP42 which encodes a core subunit of the Arabidopsis exosome and presented evidence that RRP42 is essential for the development of female gametophytes. Next, we designed three different amiRNAs targeting RRP42 . The rrp42 knock-down mutants mainly displayed variegated and serrated leaves, especially in cauline leaves. The internal anatomy of cauline leaves displayed irregularly shaped palisade cells and a reduced density of mesophyll cells. Interestingly, we detected highly accumulated mRNAs that encode xyloglucan endotransglucosylase/hydrolases (XTHs) and expansins (EXPAs) during later growth stages in rrp42 knock-down mutants. The mRNA decay kinetics analysis for XTH19 , EXPA10 , and EXPA11 revealed that RRP42 had a role in the decay of these mRNAs in the cytoplasm. RRP42 is localized to both the nucleus and cytoplasm, and RRP42 is preferentially expressed in cauline leaves during later growth stages. Altogether, our results demonstrate that RRP42 is essential for the development of female gametophytes and plays an important role in mesophyll cell morphogenesis.

Molecular cloning of a gene encoding translation initiation factor (TIF) from Candida albicans.

PubMed

Mirbod, F; Nakashima, S; Kitajima, Y; Ghannoum, M A; Cannon, R D; Nozawa, Y

1996-01-01

The differential display technique was applied to compare mRNAs from two clinical isolates of Candida albicans with different virulence; high (potent strain, 16240) and low (weak strain, 18084) extracellular phospholipase activities. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. A complementary DNA fragment seen distinctly in the potent phospholipase producing strain was highly homologous to the yeast translation initiation factor (TIF). The selected DNA fragment was then used as a probe to isolate its corresponding complementary DNA clone from a library of C. albicans genomic DNA. The sequence of isolated gene revealed an open reading frame of 1194 nucleotides with the potential to encode a protein of 397 amino acids with a predicted molecular weight of 43 kDa. Over its entire length, the amino acid sequence showed strong homology (78-89%) to Saccharomyces cerevisiae TIF and (63-80%) to mouse eIF-4A proteins. Therefore, our C. albicans gene was identified to be TIF (Ca TIF). Northern blot analysis in the two strains of C. albicans revealed that Ca TIF expression is 1.5-fold higher in the potent phospholipase producing strain. The restriction endonuclease digestion of genomic DNA from this potent strain revealed at least two hybridized bands in Southern blot analysis, suggesting two or more closely related sequences in the C. albicans genome.

Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens

PubMed Central

Möller, Philip; Overlöper, Aaron; Förstner, Konrad U.; Wen, Tuan-Nan; Sharma, Cynthia M.; Lai, Erh-Min; Narberhaus, Franz

2014-01-01

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens. PMID:25330313

Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens.

PubMed

Möller, Philip; Overlöper, Aaron; Förstner, Konrad U; Wen, Tuan-Nan; Sharma, Cynthia M; Lai, Erh-Min; Narberhaus, Franz

2014-01-01

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, B.L.; Samuel, C.E.

1985-05-01

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12more » is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.« less

Autocrine regulation of human sperm motility by tachykinins

PubMed Central

2010-01-01

Background We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Methods Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). Results The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). Conclusion These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins. PMID:20796280

Autocrine regulation of human sperm motility by tachykinins.

PubMed

Pinto, Francisco M; Ravina, Cristina G; Subiran, Nerea; Cejudo-Román, Antonio; Fernández-Sánchez, Manuel; Irazusta, Jon; Garrido, Nicolas; Candenas, Luz

2010-08-26

We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

mRNA quality control is bypassed for immediate export of stress-responsive transcripts.

PubMed

Zander, Gesa; Hackmann, Alexandra; Bender, Lysann; Becker, Daniel; Lingner, Thomas; Salinas, Gabriela; Krebber, Heike

2016-12-12

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67-Mtr2 (TAP-p15 in humans). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.

Selenium. Role of the Essential Metalloid in Health

PubMed Central

Kurokawa, Suguru; Berry, Marla J.

2015-01-01

Selenium is an essential micronutrient in mammals, but is also recognized as toxic in excess. It is a non-metal with properties that are intermediate between the chalcogen elements sulfur and tellurium. Selenium exerts its biological functions through selenoproteins. Selenoproteins contain selenium in the form of the 21st amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur-containing side chain replaced by a Se-containing side chain. Sec is encoded by the codon UGA, which is one of three termination codons for mRNA translation in non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by specialized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome. Most of the selenoprotein genes were discovered by bioinformatics approaches, searching for SECIS elements downstream of in-frame UGA codons. Sec has been described as having stronger nucleophilic and electrophilic properties than cysteine, and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins, whose functions are known, are involved in redox systems and signaling pathways. However, several selenoproteins are not well characterized in terms of their function. The selenium field has grown dramatically in the last few decades, and research on selenium biology is providing extensive new information regarding its importance for human health. PMID:24470102

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

PubMed Central

Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Identifying mRNA sequence elements for target recognition by human Argonaute proteins

PubMed Central

Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

2014-01-01

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

Genome-Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells

PubMed Central

Gu, Wei; Cui, Yizhi; Zhong, Jiayong; Jin, Jingjie; He, Qing-Yu; Wang, Tong; Zhang, Gong

2016-01-01

In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality. PMID:26926465

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation.

PubMed

Di Francesco, Andrea; Di Germanio, Clara; Panda, Amaresh C; Huynh, Phu; Peaden, Robert; Navas-Enamorado, Ignacio; Bastian, Paul; Lehrmann, Elin; Diaz-Ruiz, Alberto; Ross, David; Siegel, David; Martindale, Jennifer L; Bernier, Michel; Gorospe, Myriam; Abdelmohsen, Kotb; de Cabo, Rafael

2016-10-01

NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3' untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3'UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as an RNA-binding protein may help to explain its pleiotropic biological effects. Published by Elsevier Inc.

Modulated Expression of Genes Encoding Estrogen Metabolizing Enzymes by G1-Phase Cyclin-Dependent Kinases 6 and 4 in Human Breast Cancer Cells

PubMed Central

Jia, Yi; Domenico, Joanne; Swasey, Christina; Wang, Meiqin; Gelfand, Erwin W.; Lucas, Joseph J.

2014-01-01

G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers. PMID:24848372

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

PubMed

Tee, Meng Kian; Speek, Mart; Legeza, Balázs; Modi, Bhavi; Teves, Maria Eugenia; McAllister, Janette M; Strauss, Jerome F; Miller, Walter L

2016-10-15

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells.

PubMed

Le Moignic, A; Malard, V; Benvegnu, T; Lemiègre, L; Berchel, M; Jaffrès, P-A; Baillou, C; Delost, M; Macedo, R; Rochefort, J; Lescaille, G; Pichon, C; Lemoine, F M; Midoux, P; Mateo, V

2018-05-28

Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Their proper delivery to dendritic cells (DCs) requires their protection against RNase degradation and more specificity for dose reduction. Lipid-Polymer-RNA lipopolyplexes (LPR) are attractive mRNA delivery systems and their equipment with mannose containing glycolipid, specific of endocytic receptors present on the membrane of DCs is a valuable strategy. In this present work, we evaluated the capacity of LPR functionalized with a tri-antenna of α-d-mannopyranoside (triMN-LPR) concerning (i) their binding to CD209/DC-SIGN and CD207/Langerin expressing cell lines, human and mouse DCs and other hematopoietic cell populations, (ii) the nature of induced immune response after in vivo immunization and (iii) their therapeutic anti-cancer vaccine efficiency. We demonstrated that triMN-LPR provided high induction of a local inflammatory response two days after intradermal injection to C57BL/6 mice, followed by the recruitment and activation of DCs in the corresponding draining lymph nodes. This was associated with skin production of CCR7 and CXCR4 at vaccination sites driving DC migration. High number of E7-specific T cells was detected after E7-encoded mRNA triMN-LPR vaccination. When evaluated in three therapeutic pre-clinical murine tumor models such as E7-expressing TC1 cells, OVA-expressing EG7 cells and MART-1-expressing B16F0 cells, triMN-LPR carrying mRNA encoding the respective antigens significantly exert curative responses in mice vaccinated seven days after initial tumor inoculation. These results provide evidence that triMN-LPR give rise to an efficient stimulatory immune response allowing for therapeutic anti-cancer vaccination in mice. This mRNA formulation should be considered for anti-cancer vaccination in Humans. Copyright © 2018 Elsevier B.V. All rights reserved.

Translational control of aberrant stress responses as a hallmark of cancer.

PubMed

El-Naggar, Amal M; Sorensen, Poul H

2018-04-01

Altered mRNA translational control is emerging as a critical factor in cancer development and progression. Targeting specific elements of the translational machinery, such as mTORC1 or eIF4E, is emerging as a new strategy for innovative cancer therapy. While translation of most mRNAs takes place through cap-dependent mechanisms, a sub-population of cellular mRNA species, particularly stress-inducible mRNAs with highly structured 5'-UTR regions, are primarily translated through cap-independent mechanisms. Intriguingly, many of these mRNAs encode proteins that are involved in tumour cell adaptation to microenvironmental stress, and thus linked to aggressive behaviour including tumour invasion and metastasis. This necessitates a rigorous search for links between microenvironmental stress and aggressive tumour phenotypes. Under stress, cells block global protein synthesis to preserve energy while maintaining selective synthesis of proteins that support cell survival. One highly conserved mechanism to regulate protein synthesis under cell stress is to sequester mRNAs into cytosolic aggregates called stress granules (SGs), where their translation is silenced. SGs confer survival advantages and chemotherapeutic resistance to tumour cells under stress. Recently, it has been shown that genetically blocking SG formation dramatically reduces tumour invasive and metastatic capacity in vivo. Therefore, targeting SG formation might represent a potential treatment strategy to block cancer metastasis. Here, we present the critical link between selective mRNA translation, stress adaptation, SGs, and tumour progression. Further, we also explain how deciphering mechanisms of selective mRNA translation occurs under cell stress holds great promise for the identification of new targets in the treatment of cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression of mRNAs encoding dopamine receptors in striatal regions is differentially regulated by midbrain and hippocampal neurons.

PubMed

Brené, S; Herrera-Marschitz, M; Persson, H; Lindefors, N

1994-02-01

The glutamate analogue kainic acid was injected into the hippocampus of intact or 6-hydroxydopamine deafferented rats to investigate the influence of hippocampal neurons on the expression of dopamine D1 and D2 receptor mRNAs in subregions of the striatal complex and possible modulation by dopaminergic neurons. Quantitative in situ hybridization using 35S-labeled oligonucleotide probes specific for dopamine D1 and D2 receptor mRNAs, respectively, were used. It was found that an injection of kainic acid into the hippocampal formation had alone no significant effect on dopamine D1 or D2 receptor mRNA levels in any of the analyzed striatal subregions in animals analyzed 4 h after the injections. Kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion produced an increase in D1 receptor mRNA levels in the ipsilateral medial caudate-putamen, and a bilateral increase in core and shell of nucleus accumbens (ventral striatal limbic regions). A unilateral 6-hydroxydopamine lesion alone caused an increase in D2 receptor mRNA in the lateral caudate-putamen (dorsal striatal motor region) ipsilateral to the lesion and an increase in D1 receptor mRNA in the accumbens core ipsilateral to the lesion. However, in dopamine-lesioned animals, dopamine D1 receptor mRNA levels were increased bilaterally in nucleus accumbens core and shell and in the ipsilateral medial caudate-putamen following kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion. These results indicate a differential regulation of the expression of dopamine D1 and D2 receptor mRNAs by midbrain and hippocampal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The oncogenic triangle of HMGA2, LIN28B and IGF2BP1 antagonizes tumor-suppressive actions of the let-7 family.

PubMed

Busch, Bianca; Bley, Nadine; Müller, Simon; Glaß, Markus; Misiak, Danny; Lederer, Marcell; Vetter, Martina; Strauß, Hans-Georg; Thomssen, Christoph; Hüttelmaier, Stefan

2016-05-05

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The oncogenic triangle of HMGA2, LIN28B and IGF2BP1 antagonizes tumor-suppressive actions of the let-7 family

PubMed Central

Busch, Bianca; Bley, Nadine; Müller, Simon; Glaß, Markus; Misiak, Danny; Lederer, Marcell; Vetter, Martina; Strauß, Hans-Georg; Thomssen, Christoph; Hüttelmaier, Stefan

2016-01-01

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3′-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3′ UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment. PMID:26917013

Lack of Change in Markers of Presynaptic Terminal Abundance Alongside Subtle Reductions in Markers of Presynaptic Terminal Plasticity in Prefrontal Cortex of Schizophrenia Patients

PubMed Central

Fung, Samantha J.; Sivagnanasundaram, Sinthuja; Shannon Weickert, Cynthia

2010-01-01

Background Reduced synaptic connectivity in frontal cortex may contribute to schizophrenia symptoms. While altered mRNA and protein expression of various synaptic genes has been found, discrepancies between studies mean a generalisable synaptic pathology in schizophrenia has not been identified. Methods We determined if mRNAs encoding presynaptic proteins enriched in inhibitory [vesicular GABA transporter (VGAT) and complexin 1] and/or excitatory [vesicular glutamate transporter (VGluT1) and complexin 2] terminals are altered in the dorsolateral prefrontal cortex of subjects with schizophrenia (n=37 patients, n=37 controls). We also measured mRNA expression of markers associated with synaptic plasticity/neurite outgrowth [growth associated protein 43 (GAP43) and neuronal navigators 1 and 2 (NAV1 and NAV2)]; and mRNAs of other synaptic-associated proteins previously implicated in schizophrenia: dysbindin and vesicle-associated membrane protein (VAMP1) mRNAs using quantitative RT-PCR. Results No significant changes in complexin 1, VGAT, complexin 2, VGluT1, dysbindin, NAV2, or VAMP1 mRNA expression were found, however we observed reduced expression of mRNAs associated with plasticity/cytoskeletal modification (GAP43 and NAV1) in schizophrenia. Although dysbindin mRNA did not differ in schizophrenia compared to controls, dysbindin mRNA positively correlated with GAP-43 and NAV1 in schizophrenia, but not in controls, suggesting low levels of dysbindin may be linked to reduced plasticity in the disease state. No relationships between three dysbindin genetic polymorphisms previously associated with dysbindin mRNA levels were found. Conclusions A reduction in the plasticity of synaptic terminals supports the hypothesis that reduced modifiability of synaptic terminals may contribute to neuropathology and working memory deficits in schizophrenia. PMID:21145444

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

PubMed

Vaccaro, M C; Wilding, M; Dale, B; Campanella, C; Carotenuto, R

2012-08-01

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

PubMed Central

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination.

PubMed

Snedden, Donald D; Bertke, Michelle M; Vernon, Dominic; Huber, Paul W

2013-07-01

The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.

Genome wide responses of murine lungs to dietary α-tocopherol

PubMed Central

Oommen, Saji; Vasu, Vihas T.; Leonard, Scott W.; Traber, Maret G.; Cross, Carroll E.; Gohil, Kishorchandra

2009-01-01

α-tocopherol (α-T) may affect biological processes by modulating mRNA concentrations. This study screened the responses of ~15,000 lung mRNAs to dietary α-T in mice. The lung was chosen as the target organ because it is subjected to cyclical variations in oxidant and inflammatory stressors and α-T has been implicated in their modulations. The analysis identified ~400 mRNAs sensitive to α-T status of lungs determined by dietary α-T. The female lung transcriptome appears to be more sensitive to the α-T status than that of the male lungs. Here, we focus on the induction of 13 cytoskeleton genes by dietary α-T because they were similarly induced in the male and the female lungs. Their inductions were confirmed by quantitative-real-time-polymerase chain reaction (qRT-PCR). Immunohistochemical analyses of three of the encoded proteins suggest that they are expressed in lung vasculature and alveolar regions. The data suggest that the lung α-T status may modulate cytoarchitecture of lungs. PMID:17164183

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

PubMed

Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms.

PubMed

Ma, Xing; Zhu, Xiujuan; Han, Yingying; Story, Benjamin; Do, Trieu; Song, Xiaoqing; Wang, Su; Zhang, Ying; Blanchette, Marco; Gogol, Madelaine; Hall, Kate; Peak, Allison; Anoja, Perera; Xie, Ting

2017-04-24

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. Here we report that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors. Copyright © 2017 Elsevier Inc. All rights reserved.

Discriminating tastes

PubMed Central

Storz, Gisela

2011-01-01

Hfq-binding small RNAs (sRNAs) are critical regulators that form limited base-pairing interactions with target mRNAs in bacteria. These sRNAs have been linked to diverse environmental responses, yet little is known how Hfq-binding sRNAs participate in the regulatory networks associated with each response. We recently described how the Hfq-binding sRNA Spot 42 in Escherichia coli contributes to catabolite repression, a regulatory phenomenon that allows bacteria to consume some carbon sources over others. Spot 42 base pairs with numerous mRNAs encoding enzymes in central and secondary metabolism, redox balancing, and the uptake and consumption of non-preferred carbon sources. Many of the corresponding genes are transcriptionally activated by the Spot 42-repressor CRP, forming a regulatory circuit called a multi-output feedforward loop. We found that this loop influences both the steady-state levels and dynamics of gene regulation. In this article, we discuss how the CRP-Spot 42 feedforward loop is integrated into encompassing networks and how this loop may benefit enteric bacteria facing uncertain and changing nutrient conditions. PMID:21788732

Regulation of rice root development by a retrotransposon acting as a microRNA sponge.

PubMed

Cho, Jungnam; Paszkowski, Jerzy

2017-08-26

It is well documented that transposable elements (TEs) can regulate the expression of neighbouring genes. However, their ability to act in trans and influence ectopic loci has been reported rarely. We searched in rice transcriptomes for tissue-specific expression of TEs and found them to be regulated developmentally. They often shared sequence homology with co-expressed genes and contained potential microRNA-binding sites, which suggested possible contributions to gene regulation. In fact, we have identified a retrotransposon that is highly transcribed in roots and whose spliced transcript constitutes a target mimic for miR171. miR171 destabilizes mRNAs encoding the root-specific family of SCARECROW-Like transcription factors. We demonstrate that retrotransposon-derived transcripts act as decoys for miR171, triggering its degradation and thus results in the root-specific accumulation of SCARECROW-Like mRNAs. Such transposon-mediated post-transcriptional control of miR171 levels is conserved in diverse rice species.

Separate analysis of human papillomavirus E6 and E7 messenger RNAs to predict cervical neoplasia progression

PubMed Central

Liu, Shuling; Lachkar, Bouchra; Zhang, Shuang; Xu, Chenyang; Tenjimbayashi, Yuri; Shikama, Ayumi; Tasaka, Nobutaka; Akiyama, Azusa; Sakurai, Manabu; Nakao, Sari; Ochi, Hiroyuki; Onuki, Mamiko; Matsumoto, Koji; Yoshikawa, Hiroyuki; Satoh, Toyomi

2018-01-01

A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA (p = 0.00011, 0.80 and 0.54). The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA (p = 0.034, 0.73, 0.53, and 0.72). Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings. PMID:29466435

Identification of human microRNA targets from isolated argonaute protein complexes.

PubMed

Beitzinger, Michaela; Peters, Lasse; Zhu, Jia Yun; Kremmer, Elisabeth; Meister, Gunter

2007-06-01

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

PubMed

Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

2017-05-01

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes

PubMed Central

Arikawa, Kentaro; Iwanaga, Tomoyuki; Wakakuwa, Motohiro; Kinoshita, Michiyo

2017-01-01

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved. PMID:29238294

Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

PubMed Central

Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I.; Beyer, Richard P.; Gray, Lucas T.; Welsh, Judith A.; Schetter, Aaron J.; Kumamoto, Kensuke; Wang, Xin Wei; Hickson, Ian D.; Maizels, Nancy; Monnat, Raymond J.; Harris, Curtis C.

2014-01-01

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome. PMID:24958861

Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs.

PubMed

Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I; Beyer, Richard P; Gray, Lucas T; Welsh, Judith A; Schetter, Aaron J; Kumamoto, Kensuke; Wang, Xin Wei; Hickson, Ian D; Maizels, Nancy; Monnat, Raymond J; Harris, Curtis C

2014-07-08

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.

A viral microRNA functions as an ortholog of cellular miR-155

PubMed Central

Gottwein, Eva; Mukherjee, Neelanjan; Sachse, Christoph; Frenzel, Corina; Majoros, William H.; Chi, Jen-Tsan A.; Braich, Ravi; Manoharan, Muthiah; Soutschek, Jürgen; Ohler, Uwe; Cullen, Bryan R.

2008-01-01

All metazoan eukaryotes express microRNAs (miRNAs), ∼22 nt regulatory RNAs that can repress the expression of mRNAs bearing complementary sequences1. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis2. While specific viral miRNAs have been shown to autoregulate viral mRNAs3,4 or downregulate cellular mRNAs5,6, the function of the majority of viral miRNAs remains unknown. Here, we report that the miR-K12−11 miRNA encoded by Kaposi's Sarcoma Associated Herpesvirus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA “seed” region7. Using a range of assays, we demonstrate that expression of physiological levels of miR-K12−11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12−11 functions as an ortholog of cellular miR-155 and has likely evolved to exploit a pre-existing gene regulatory pathway in B-cells. Moreover, the known etiological role of miR-155 in B-cell transformation8-10 suggests that miR-K12−11 may contribute to the induction of KSHV-positive B-cell tumors in infected patients. PMID:18075594

Gene 2 of the sigma rhabdovirus genome encodes the P protein, and gene 3 encodes a protein related to the reverse transcriptase of retroelements.

PubMed

Landès-Devauchelle, C; Bras, F; Dezélée, S; Teninges, D

1995-11-10

The nucleotide sequence of the genes 2 and 3 of the Drosophila rhabdovirus sigma was determined from cDNAs to viral genome and poly(A)+ mRNAs. Gene 2 comprises 1032 nucleotides and contains a long ORF encoding a molecular weight 35,208 polypeptide present in infected cells and in virions which migrates in SDS-PAGE as a doublet of M(r) about 60 kDa. The distribution of acidic charges as well as the electrophoretic properties of the protein are characteristic of the rhabdovirus P proteins. Gene 3 comprises 923 nucleotides and contains a long ORF capable of coding a polypeptide of 298 amino acids of MW 33,790. The putative protein (PP3) is similar in size to a minor component of the virions. Computer analysis shows that the sequence of PP3 contains three motifs related to the conserved motifs of reverse transcriptases.

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

PubMed

Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

2009-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue.

PubMed

Wang, S Y; Gudas, L J

1990-09-15

We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

RNA-seq Analysis of Clinical-Grade Osteochondral Allografts Reveals Activation of Early Response Genes

PubMed Central

Lin, Yang; Lewallen, Eric A.; Camilleri, Emily T.; Bonin, Carolina A.; Jones, Dakota L.; Dudakovic, Amel; Galeano-Garces, Catalina; Wang, Wei; Karperien, Marcel J.; Larson, Annalise N.; Dahm, Diane L.; Stuart, Michael J.; Levy, Bruce A.; Smith, Jay; Ryssman, Daniel B.; Westendorf, Jennifer J.; Im, Hee-Jeong; van Wijnen, Andre J.; Riester, Scott M.; Krych, Aaron J.

2016-01-01

Preservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA-seq) and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assess genome-wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM-related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. PMID:26909883

The phosphoprotein genes of measles viruses from subacute sclerosing panencephalitis cases encode functional as well as non-functional proteins and display reduced editing.

PubMed

Millar, E L; Rennick, L J; Weissbrich, B; Schneider-Schaulies, J; Duprex, W P; Rima, B K

2016-01-04

Products expressed from the second (P/V/C) gene are important in replication and abrogating innate immune responses during acute measles virus (MV) infection. Thirteen clone sets were derived from the P/V/C genes of measles virus (MV) RNA extracted from brains of a unique collection of seven cases of subacute sclerosing panencephalitis (SSPE) caused by persistent MV in the central nervous system (CNS). Whether these functions are fully maintained when MV replicates in the CNS has not been previously determined. Co-transcriptional editing of the P mRNAs by non-template insertion of guanine (G) nucleotides, which generates mRNAs encoding the viral V protein, occurs much less frequently (9%) in the SSPE derived samples than during the acute infection (30-50%). Thus it is likely that less V protein, which is involved in combatting the innate immune response, is produced. The P genes in MV from SSPE cases were not altered by biased hypermutation but exhibited a high degree of variation within each case. Most but not all SSPE derived phospho-(P) proteins were functional in mini genome replication/transcription assays. An eight amino acid truncation of the carboxyl-terminus made the P protein non-functional while the insertion of an additional glycine residue by insertion of G nucleotides at the editing site had no effect on protein function. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

PubMed

Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

2016-02-12

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.

PubMed

Ahmad, Muzammil; Shen, Weiping; Li, Wen; Xue, Yutong; Zou, Sige; Xu, Dongyi; Wang, Weidong

2017-03-17

Human cells contain five topoisomerases in the nucleus and cytoplasm, but which one is the major topoisomerase for mRNAs is unclear. To date, Top3β is the only known topoisomerase that possesses RNA topoisomerase activity, binds mRNA translation machinery and interacts with an RNA-binding protein, FMRP, to promote synapse formation; and Top3β gene deletion has been linked to schizophrenia. Here, we show that Top3β is also the most abundant mRNA-binding topoisomerase in cells. Top3β, but not other topoisomerases, contains a distinctive RNA-binding domain; and deletion of this domain diminishes the amount of Top3β that associates with mRNAs, indicating that Top3β is specifically targeted to mRNAs by its RNA binding domain. Moreover, Top3β mutants lacking either its RNA-binding domain or catalytic residue fail to promote synapse formation, suggesting that Top3β requires both its mRNA-binding and catalytic activity to facilitate neurodevelopment. Notably, Top3β proteins bearing point mutations from schizophrenia and autism individuals are defective in association with FMRP; whereas one of the mutants is also deficient in binding mRNAs, catalyzing RNA topoisomerase reaction, and promoting synapse formation. Our data suggest that Top3β is the major topoisomerase for mRNAs, and requires both RNA binding and catalytic activity to promote neurodevelopment and prevent mental dysfunction. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

PubMed Central

Ohno, Misa; Togashi, Yuto; Tsuda, Kyoko; Okawa, Kazuaki; Kamaya, Minori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

2013-01-01

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific. PMID:23826286

Identification of human short introns

PubMed Central

Abebrese, Emmanuel L.; Arnold, Zachary R.; Armstrong, Katharine; Burns, Lindsay; Day, R. Thomas; Hsu, Daniel G.; Jarrell, Katherine; Luo, Yi; Mugayo, Daphine

2017-01-01

Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation. PMID:28520720

The role of alternative splicing coupled to nonsense-mediated mRNA decay in human disease.

PubMed

da Costa, Paulo J; Menezes, Juliane; Romão, Luísa

2017-10-01

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

PubMed

Van Gelder, R N; Bae, H; Palazzolo, M J; Krasnow, M A

1995-12-01

Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes.

PubMed

Bour, Sandy; Daviaud, Danièle; Gres, Sandra; Lefort, Corinne; Prévot, Danielle; Zorzano, Antonio; Wabitsch, Martin; Saulnier-Blache, Jean-Sébastien; Valet, Philippe; Carpéné, Christian

2007-08-01

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.

RNA Editing Underlies Temperature Adaptation in K+ Channels from Polar Octopuses

PubMed Central

Garrett, Sandra; Rosenthal, Joshua J.C.

2014-01-01

To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here we show that delayed rectifier K+ channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed mRNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel’s pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus A-to-I RNA editing can respond to the physical environment. PMID:22223739

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

PubMed Central

Travis, G H; Sutcliffe, J G

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA. Images PMID:2894033

Alternative splicing within the ligand binding domain of the human constitutive androstane receptor.

PubMed

Savkur, Rajesh S; Wu, Yifei; Bramlett, Kelli S; Wang, Minmin; Yao, Sufang; Perkins, Douglas; Totten, Michelle; Searfoss, George; Ryan, Timothy P; Su, Eric W; Burris, Thomas P

2003-01-01

The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug-drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

The Mitochondrial Genome and Transcriptome of the Basal Dinoflagellate Hematodinium sp.: Character Evolution within the Highly Derived Mitochondrial Genomes of Dinoflagellates

PubMed Central

Gornik, S. G.; Waller, R. F.

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA. PMID:22113794

The mitochondrial genome and transcriptome of the basal dinoflagellate Hematodinium sp.: character evolution within the highly derived mitochondrial genomes of dinoflagellates.

PubMed

Jackson, C J; Gornik, S G; Waller, R F

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.

Functional relevance of three proopiomelanocortin (POMC) genes in darkening camouflage, blind-side hypermelanosis, and appetite of Paralichthys olivaceus.

PubMed

Kang, Duk-Young; Kim, Hyo-Chan

2015-01-01

To determine whether proopiomelanocortin (POMC) genes are involved in darkening color camouflage, blind-side hypermelanosis, and appetite in flatfish, we isolated and cloned three POMC genes from the pituitary of the olive flounder (Paralichthys olivaceus) and compared their amino acid (aa) structures to those of POMC genes from other animals. Next, we examined the relationship of these pituitary POMC genes to camouflage color change, blind-side hypermelanosis, and appetite by quantifying mRNA expression. Olive flounder (of)-POMC1, 2, and 3 cDNAs consisted of 648-bp, 582-bp, and 693-bp open reading frames (ORF) encoding 216 aa, 194 aa, and 231 aa residues, respectively. Structurally, the three of-POMC cDNAs consisted of seven peptides (signal peptide, N-POMC, α-MSH, CLIP, N-β-LPH, β-MSH and β-END [or END-like peptide]) that are similar to those of other fish POMC cDNAs. α-MSH encoded a protein composed of 13 aa and β-MSH encoded a protein composed of 17 aa. The three POMC genes were predominantly expressed in the pituitary gland, but they were also expressed in a variety of tissues, including brain, eye, kidney, heart, testis, and skin. of-POMC2 exhibited the highest expression, while of-POMC3 displayed the lowest expression. The relative levels of of-POMC1 and 3 mRNAs were not influenced by background color and feeding (or fasting), but the relative level of of-POMC2 mRNA significantly increased in response to a dark background and fasting. The relative levels of of-POMC1 and 2 mRNAs were significantly higher in hypermelanic fish; however, we did not determine a direct anorexigenic or orexigenic relationship for the three POMC genes. These results indicate that pituitary POMC genes are related to darkening color change and the differentiation of pigment cells, but they are not directly related to appetite. Copyright © 2014 Elsevier Inc. All rights reserved.

Dissection of Ire1 Functions Reveals Stress Response Mechanisms Uniquely Evolved in Candida glabrata

PubMed Central

Miyazaki, Taiga; Nakayama, Hironobu; Nagayoshi, Yohsuke; Kakeya, Hiroshi; Kohno, Shigeru

2013-01-01

Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata. PMID:23382685

Maternal nutrient deprivation induces sex-specific changes in thyroid hormone receptor and deiodinase expression in the fetal guinea pig brain

PubMed Central

Chan, Shiao Y; Andrews, Marcus H; Lingas, Rania; McCabe, Chris J; Franklyn, Jayne A; Kilby, Mark D; Matthews, Stephen G

2005-01-01

Thyroid hormone deprivation during fetal life has been implicated in neurodevelopmental morbidity. In humans, poor growth in utero is also associated with fetal hypothyroxinaemia. In guinea pigs, a short period (48 h) of maternal nutrient deprivation at gestational day (gd) 50 results in fetuses with hypothyroxinaemia and increased brain/body weight ratios. Thyroid hormone action is mediated by nuclear thyroid hormone receptors (TRs) and is dependent upon the prereceptor regulation of supply of triiodothyronine (T3) by deiodinase enzymes. Examination of fetal guinea pig brains using in situ hybridization demonstrated widespread expression of mRNAs encoding TRα1, α2 and β1, with regional colocalization of deiodinase type 2 (D2) mRNA in the developing forebrain, limbic structures, brainstem and cerebellum at gd52. With maternal nutrient deprivation, TRα1 and β1 mRNA expression was significantly increased in the male, but decreased in the female fetal hippocampus and cerebellum and other areas showing high TR expression under euthyroid conditions. Maternal nutrient deprivation resulted in elevated D2 mRNA expression in males and females. Deiodinase type 3 (D3) mRNA expression was confined to the shell of the nucleus accumbens, the posterior amygdalohippocampal area, brainstem and cerebellum, and did not change with maternal nutrient deprivation. In conclusion, maternal nutrient deprivation resulted in sex-specific changes in TR mRNA expression and a generalized increase in D2 mRNAs within the fetal brain. These changes may represent a protective mechanism to maintain appropriate thyroid hormone action in the face of fetal hypothyroxinaemia in order to optimize brain development. PMID:15878952

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

PubMed Central

Lioliou, Efthimia; Sharma, Cynthia M.; Caldelari, Isabelle; Helfer, Anne-Catherine; Fechter, Pierre; Vandenesch, François; Vogel, Jörg; Romby, Pascale

2012-01-01

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. PMID:22761586

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

PubMed Central

van der Vos, Kristan E.; Abels, Erik R.; Zhang, Xuan; Lai, Charles; Carrizosa, Esteban; Oakley, Derek; Prabhakar, Shilpa; Mardini, Osama; Crommentuijn, Matheus H. W.; Skog, Johan; Krichevsky, Anna M.; Stemmer-Rachamimov, Anat; Mempel, Thorsten R.; El Khoury, Joseph; Hickman, Suzanne E.; Breakefield, Xandra O.

2016-01-01

Background To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. Methods Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. Results Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. Conclusions Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs. PMID:26433199

A single phosphorodiamidate morpholino oligomer targeting VP24 protects rhesus monkeys against lethal Ebola virus infection.

PubMed

Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Heald, Alison E; Charleston, Jay S; Sazani, Pete; Reid, St Patrick; Iversen, Patrick L; Bavari, Sina

2015-02-10

Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval. Copyright © 2015 Warren et al.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

PubMed

Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

2010-05-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

Overlapping regional distribution of CCK and TPPII mRNAs in Cynomolgus monkey brain and correlated levels in human cerebral cortex (BA 10).

PubMed

Radu, Diana; Tomkinson, Birgitta; Zachrisson, Olof; Weber, Günther; de Belleroche, Jacqueline; Hirsch, Steven; Lindefors, Nils

2006-08-09

Tripeptidyl peptidase II (TPPII) is a high molecular weight exopeptidase important in inactivating extracellular cholecystokinin (CCK). Our aims were to study the anatomical localization of TPPII and CCK mRNA in the Cynomolgus monkey brain as a basis for a possible functional anatomical connection between enzyme (TPPII) and substrate (CCK) and examine if indications of changes in substrate availability in the human brain might be reflected in changes of levels of TPPII mRNA. mRNA in situ hybridization on postmortem brain from patients having had a schizophrenia diagnosis as compared to controls and on monkey and rat brain slices. overlapping distribution patterns of mRNAs for TPPII and CCK in rat and monkey. High amounts of TPPII mRNA are seen in the neocortex, especially in the frontal region and the hippocampus. TPPII mRNA is also present in the basal ganglia and cerebellum where CCK immunoreactivity and/or CCK B receptors have been found in earlier studies, suggesting presence of CCK-ergic afferents from other brain regions. Levels of mRNAs for CCK and TPPII show a positive correlation in postmortem human cerebral cortex Brodmann area (BA) 10. TPPII mRNA might be affected following schizophrenia. overall TPPII and CCK mRNA show a similar distribution in rat and monkey brain, confirming and extending earlier studies in rodents. In addition, correlated levels of TPPII and CCK mRNA in human BA 10 corroborate a functional link between CCK and TPPII in the human brain.

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.

PubMed

Sanford, Jeremy R; Wang, Xin; Mort, Matthew; Vanduyn, Natalia; Cooper, David N; Mooney, Sean D; Edenberg, Howard J; Liu, Yunlong

2009-03-01

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.

Molecular characterization of a family of ligands for eph-related tyrosine kinase receptors.

PubMed Central

Beckmann, M P; Cerretti, D P; Baum, P; Vanden Bos, T; James, L; Farrah, T; Kozlosky, C; Hollingsworth, T; Shilling, H; Maraskovsky, E

1994-01-01

A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligand's mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph-related receptor molecules, we refer to these ligands as LERKs (ligands for eph-related kinases). Images PMID:8070404

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

PubMed Central

Wissing, Silke; Muñoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2012-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

MicroRNAs and cancer.

PubMed

Cowland, Jack B; Hother, Christoffer; Grønbaek, Kirsten

2007-10-01

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

PubMed

Sumiyoshi, Miho; Sato, Seiji; Takeda, Yukimasa; Sumida, Kazunori; Koga, Keita; Itoh, Tsunao; Nakagawa, Hiroyuki; Shimohigashi, Yasuyuki; Shimohigashi, Miki

2011-12-01

Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain.

RNA Nuclear Export: From Neurological Disorders to Cancer.

PubMed

Hautbergue, Guillaume M

2017-01-01

The presence of a nuclear envelope, also known as nuclear membrane, defines the structural framework of all eukaryotic cells by separating the nucleus, which contains the genetic material, from the cytoplasm where the synthesis of proteins takes place. Translation of proteins in Eukaryotes is thus dependent on the active transport of DNA-encoded RNA molecules through pores embedded within the nuclear membrane. Several mechanisms are involved in this process generally referred to as RNA nuclear export or nucleocytoplasmic transport of RNA. The regulated expression of genes requires the nuclear export of protein-coding messenger RNA molecules (mRNAs) as well as non-coding RNAs (ncRNAs) together with proteins and pre-assembled ribosomal subunits. The nuclear export of mRNAs is intrinsically linked to the co-transcriptional processing of nascent transcripts synthesized by the RNA polymerase II. This functional coupling is essential for the survival of cells allowing for timely nuclear export of fully processed transcripts, which could otherwise cause the translation of abnormal proteins such as the polymeric repeat proteins produced in some neurodegenerative diseases. Alterations of the mRNA nuclear export pathways can also lead to genome instability and to various forms of cancer. This chapter will describe the molecular mechanisms driving the nuclear export of RNAs with a particular emphasis on mRNAs. It will also review their known alterations in neurological disorders and cancer, and the recent opportunities they offer for the potential development of novel therapeutic strategies.

Kinases of eIF2a Switch Translation of mRNA Subset during Neuronal Plasticity

PubMed Central

Chesnokova, Ekaterina; Bal, Natalia

2017-01-01

Compared to other types of cells, neurons express the largest number of diverse mRNAs, including neuron-specific ones. This mRNA diversity is required for neuronal function, memory storage, maintenance and retrieval. Regulation of translation in neurons is very complicated and involves various proteins. Some proteins, implementing translational control in other cell types, are used by neurons for synaptic plasticity. In this review, we discuss the neuron-specific activity of four kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), general control nonderepressible 2 kinase (GCN2), and heme-reguated eIF2α kinase (HRI), the substrate for which is α-subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α is necessary for the cell during stress conditions, such as lack of amino acids, energy stress or viral infection. We propose that, during memory formation, neurons use some mechanisms similar to those involved in the cellular stress. The four eIF2α kinases regulate translation of certain mRNAs containing upstream open reading frames (uORFs). These mRNAs encode proteins involved in the processes of long-term potentiation (LTP) or long-term depression (LTD). The review examines some neuronal proteins for which translation regulation by eIF2 was suggested and checked experimentally. Of such proteins, we pay close attention to protein kinase Mζ, which is involved in memory storage and regulated at the translational level. PMID:29065505

Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

PubMed Central

Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

2011-01-01

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood

PubMed Central

Peters, Oliver; Rajewsky, Nikolaus

2015-01-01

Covalently closed circular RNA molecules (circRNAs) have recently emerged as a class of RNA isoforms with widespread and tissue specific expression across animals, oftentimes independent of the corresponding linear mRNAs. circRNAs are remarkably stable and sometimes highly expressed molecules. Here, we sequenced RNA in human peripheral whole blood to determine the potential of circRNAs as biomarkers in an easily accessible body fluid. We report the reproducible detection of thousands of circRNAs. Importantly, we observed that hundreds of circRNAs are much higher expressed than corresponding linear mRNAs. Thus, circRNA expression in human blood reveals and quantifies the activity of hundreds of coding genes not accessible by classical mRNA specific assays. Our findings suggest that circRNAs could be used as biomarker molecules in standard clinical blood samples. PMID:26485708

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

PubMed Central

de Souza, Sandro J.; Camargo, Anamaria A.; Briones, Marcelo R. S.; Costa, Fernando F.; Nagai, Maria Aparecida; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; de Fátima Sonati, Maria; Tajara, Eloiza H.; Valentini, Sandro R.; Acencio, Marcio; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Bengtson, Mário Henrique; Carraro, Dirce M.; Carvalho, Alex F.; Carvalho, Lúcia Helena; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Costa, Maria Cristina R.; Curcio, Cyntia; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Leite, Luciana C. C.; Maia, Gustavo; Majumder, Paromita; Marins, Mozart; Matsukuma, Adriana; Melo, Analy S. A.; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana Gilbert; Rahal, Paula; Rainho, Claudia A.; da Ro's, Nancy; de Sá, Renata G.; Sales, Magaly M.; da Silva, Neusa P.; Silva, Tereza C.; da Silva, Wilson; Simão, Daniel F.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Zalcberg, Heloisa; Brentani, Ricardo R.; Reis, Luis F. L.; Dias-Neto, Emmanuel; Simpson, Andrew J. G.

2000-01-01

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html). PMID:11070084

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

PubMed

Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Deadenylase depletion protects inherited mRNAs in primordial germ cells

PubMed Central

Swartz, S. Zachary; Reich, Adrian M.; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M.; Campanale, Joseph P.; Hamdoun, Amro; Wessel, Gary M.

2014-01-01

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a ‘time capsule’ model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. PMID:25100654

RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation.

PubMed

Nakayama, Kei; Ohashi, Rie; Shinoda, Yo; Yamazaki, Maya; Abe, Manabu; Fujikawa, Akihiro; Shigenobu, Shuji; Futatsugi, Akira; Noda, Masaharu; Mikoshiba, Katsuhiko; Furuichi, Teiichi; Sakimura, Kenji; Shiina, Nobuyuki

2017-11-21

Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation.

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5.

PubMed

Nuñez, Illyce; Rodriguez Pino, Marbelys; Wiley, David J; Das, Maitreyi E; Chen, Chuan; Goshima, Tetsuya; Kume, Kazunori; Hirata, Dai; Toda, Takashi; Verde, Fulvia

2016-07-30

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Salt-loading increases vasopressin and vasopressin 1b receptor mRNA in the hypothalamus and choroid plexus.

PubMed

Zemo, D A; McCabe, J T

2001-01-01

The choroid plexus plays a pivotal role in the production of cerebrospinal fluid (CSF). Messenger RNA (mRNA) transcripts encoding arginine vasopressin (AVP) and the vasopressin 1b receptor (V(1b)R) are found in various structures of the central nervous system, including the choroid plexus. The present study measured AVP and V(1b)R mRNA production in response to plasma hyperosmolality. Compared to rats maintained on water, 2% salt-drinking rats had increased levels of AVP and V(1b)R mRNAs in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and in the choroid plexus. The increase in V(1b)R mRNA in the SON and PVN as a result of plasma hyperosmolality may reflect changes in receptor production that, in turn, have a role in AVP autoregulation of hypothalamic magnocellular neurons. The increase of AVP and V(1b)R mRNAs in the choroid plexus further shows the involvement of AVP in the regulation of brain water content and cerebral edema. Copyright 2001 Harcourt Publishers Ltd.

A multigene locus containing the Manx and bobcat genes is required for development of chordate features in the ascidian tadpole larva.

PubMed

Swalla, B J; Just, M A; Pederson, E L; Jeffery, W R

1999-04-01

The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5' untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5' end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat mRNA accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta X M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.

Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression

PubMed Central

Sullivan, Eileen; Santiago, Carlos; Parker, Emily D.; Dominski, Zbigniew; Yang, Xiaocui; Lanzotti, David J.; Ingledue, Tom C.; Marzluff, William F.; Duronio, Robert J.

2001-01-01

Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem–loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3′ end. Stem–loop–binding protein (SLBP) binds to the histone mRNA 3′ end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3′ ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3′ end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle. PMID:11157774

Genes encoding major light-harvesting polypeptides are clustered on the genome of the cyanobacterium Fremyella diplosiphon.

PubMed Central

Conley, P B; Lemaux, P G; Lomax, T L; Grossman, A R

1986-01-01

The polypeptide composition of the phycobilisome, the major light-harvesting complex of prokaryotic cyanobacteria and certain eukaryotic algae, can be modulated by different light qualities in cyanobacteria exhibiting chromatic adaptation. We have identified genomic fragments encoding a cluster of phycobilisome polypeptides (phycobiliproteins) from the chromatically adapting cyanobacterium Fremyella diplosiphon using previously characterized DNA fragments of phycobiliprotein genes from the eukaryotic alga Cyanophora paradoxa and from F. diplosiphon. Characterization of two lambda-EMBL3 clones containing overlapping genomic fragments indicates that three sets of phycobiliprotein genes--the alpha- and beta-allophycocyanin genes plus two sets of alpha- and beta-phycocyanin genes--are clustered within 13 kilobases on the cyanobacterial genome and transcribed off the same strand. The gene order (alpha-allophycocyanin followed by beta-allophycocyanin and beta-phycocyanin followed by alpha-phycocyanin) appears to be a conserved arrangement found previously in a eukaryotic alga and another cyanobacterium. We have reported that one set of phycocyanin genes is transcribed as two abundant red light-induced mRNAs (1600 and 3800 bases). We now present data showing that the allophycocyanin genes and a second set of phycocyanin genes are transcribed into major mRNAs of 1400 and 1600 bases, respectively. These transcripts are present in RNA isolated from cultures grown in red and green light, although lower levels of the 1600-base phycocyanin transcript are present in cells grown in green light. Furthermore, a larger transcript of 1750 bases hybridizes to the allophycocyanin genes and may be a precursor to the 1400-base species. Images PMID:3086870

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

PubMed

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Van Seuningen, Isabelle; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-03-06

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3 -/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor–activated macrophages

PubMed Central

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-01-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages. PMID:22675026

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

PubMed

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-08-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

PubMed Central

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-01-01

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

Tristetraprolin regulates the expression of the human inducible nitric-oxide synthase gene.

PubMed

Fechir, Marcel; Linker, Katrin; Pautz, Andrea; Hubrich, Thomas; Förstermann, Ulrich; Rodriguez-Pascual, Fernando; Kleinert, Hartmut

2005-06-01

The expression of human inducible NO synthase (iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogen-activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominant-negative p38 MAPKalpha protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, quantitative real-time polymerase chain reaction, and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580 incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP), which is known to recruit mRNAs containing AU-rich elements to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580 treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Because tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms

PubMed Central

King, Elizabeth M.; Chivers, Joanna E.; Rider, Christopher F.; Minnich, Anne; Giembycz, Mark A.; Newton, Robert

2013-01-01

Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the de novo synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent repression by dexamethasone. In conclusion, our data indicate roles for both transrepression and transactivation in the glucocorticoid-dependent repression of inflammatory gene expression. However, transactivation appears to account for the more potent and efficacious mechanism of repression by glucocorticoids on these IL-1β-induced genes. PMID:23349769

T cell post-transcriptional miRNA-mRNA interaction networks identify targets associated with susceptibility/resistance to collagen-induced arthritis.

PubMed

Donate, Paula B; Fornari, Thais A; Macedo, Claudia; Cunha, Thiago M; Nascimento, Daniele C B; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Cunha, Fernando Q; Passos, Geraldo A

2013-01-01

Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

Characterization and differential expression of three GnRH forms during reproductive development in cultured turbot Schophthalmus maximus

NASA Astrophysics Data System (ADS)

Zhao, Chunyan; Xu, Shihong; Feng, Chengcheng; Liu, Yifan; Yang, Yang; Wang, Yanfeng; Xiao, Yongshuang; Song, Zongcheng; Liu, Qinghua; Li, Jun

2017-10-01

Turbots (Schophthalmus maximus), one of the most important economic marine flatfish species, fail to undergo final spawning and spermiation naturally under artificial farming conditions. In vertebrates, reproduction is regulated by the brain-pituitary-gonadal axis (BPG-axis), and gonadotropin releasing hormone (GnRH) is one of its key components. Therefore, to better understand the physiology of reproduction in the turbot, three of the genes encoding GnRH subtypes—sbGnRH, cGnRH-II and sGnRH—were cloned and sequenced by isolating the cDNA sequences. The localizations and patterns of expression of their mRNAs were also evaluated during seasonal gonadal development. All three mRNAs were expressed abundantly in the brain; sbGnRH and sGnRH mRNAs were also detected in the gonads and pituitary gland, and sbGnRH expression was much higher than that of sGnRH, indicating the critical role of sbGnRH in regulating the BPG-axis. Moreover, the brain expression patterns of sbGnRH and sGnRH mRNAs showed an increased trend during gonadal development, peaking in mature stages. This indicated the direct regulation of gonadal development by the GnRH system. In addition, cGnRH-II mRNA expression showed no significant variations, suggesting that cGnRH-II is not critically involved in the control of reproduction. Further, the mRNA abundances of the three GnRH forms in the breeding season were significantly higher than those in immature and post-breeding stages in all analyzed brain areas. Therefore, we propose that sbGnRH is the most important hormone for the regulation of reproduction in turbot via the BPG-axis. These results will help in better understanding the reproductive endocrine mechanisms of turbots and lay the groundwork for additional studies aimed at comparing the reproductive physiology of wild individuals with those raised under artificial conditions.

HuR binding to cytoplasmic mRNA is perturbed by heat shock

PubMed Central

Gallouzi, Imed-Eddine; Brennan, Christopher M.; Stenberg, Myrna G.; Swanson, Maurice S.; Eversole, Ashley; Maizels, Nancy; Steitz, Joan A.

2000-01-01

AU-rich elements (AREs) located in the 3′ untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37°C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)+ RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12–15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)+ RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins. PMID:10737787

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

PubMed Central

Zubiaga, A M; Belasco, J G; Greenberg, M E

1995-01-01

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process. PMID:7891716

A Novel Hybrid Iron Regulation Network Combines Features from Pathogenic and Nonpathogenic Yeasts

PubMed Central

Gerwien, Franziska; Safyan, Abu; Wisgott, Stephanie; Hille, Fabrice; Kaemmer, Philipp; Linde, Jörg; Brunke, Sascha; Kasper, Lydia

2016-01-01

ABSTRACT Iron is an essential micronutrient for both pathogens and their hosts, which restrict iron availability during infections in an effort to prevent microbial growth. Successful human pathogens like the yeast Candida glabrata have thus developed effective iron acquisition strategies. Their regulation has been investigated well for some pathogenic fungi and in the model organism Saccharomyces cerevisiae, which employs an evolutionarily derived system. Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi. Specifically, similarly to baker’s yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes. However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens. Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi. PMID:27795405

Analysis of Ribosome Stalling and Translation Elongation Dynamics by Deep Learning.

PubMed

Zhang, Sai; Hu, Hailin; Zhou, Jingtian; He, Xuan; Jiang, Tao; Zeng, Jianyang

2017-09-27

Ribosome stalling is manifested by the local accumulation of ribosomes at specific codon positions of mRNAs. Here, we present ROSE, a deep learning framework to analyze high-throughput ribosome profiling data and estimate the probability of a ribosome stalling event occurring at each genomic location. Extensive validation tests on independent data demonstrated that ROSE possessed higher prediction accuracy than conventional prediction models, with an increase in the area under the receiver operating characteristic curve by up to 18.4%. In addition, genome-wide statistical analyses showed that ROSE predictions can be well correlated with diverse putative regulatory factors of ribosome stalling. Moreover, the genome-wide ribosome stalling landscapes of both human and yeast computed by ROSE recovered the functional interplays between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particles and protein secondary structure formation. Overall, our study provides a novel method to complement the ribosome profiling techniques and further decipher the complex regulatory mechanisms underlying translation elongation dynamics encoded in the mRNA sequence. Copyright © 2017 Elsevier Inc. All rights reserved.

Bromelain Treatment Decreases Secretion of Pro-Inflammatory Cytokines and Chemokines by Colon Biopsies In Vitro

PubMed Central

Onken, Jane E.; Greer, Paula K.; Calingaert, Brian; Hale, Laura P.

2008-01-01

Oral bromelain has been anecdotally reported to decrease inflammation in ulcerative colitis (UC). Proteolytically active bromelain is known to decrease expression of mRNAs encoding pro-inflammatory cytokines by human leukocytes in vitro. To assess the effect of bromelain on mucosal secretion of cytokines in inflammatory bowel disease (IBD), endoscopic colon biopsies from patients with UC, Crohn’s disease (CD), and non-IBD controls were treated in vitro with bromelain or media, then cultured. Secretion of pro-inflammatory cytokines and chemokines was measured. Significant increases in granulocyte colony stimulating factor (G-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) were detected in the media from actively inflamed areas in UC and CD as compared with non-inflamed IBD tissue and non-IBD controls. In vitro bromelain treatment decreased secretion of G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-γ, CCL4/macrophage inhibitory protein (MIP)-1β, and TNF by inflamed tissue in IBD. Bromelain may be a novel therapy for IBD. PMID:18160345

Modulation of TLR2 protein expression by a miR-105 in human oral keratinocytes

EPA Science Inventory

Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and can regulate innate immune responses. Our model system comprised primary human keratinoc...

DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karathanasis, S.K.; Ferris, E.; Haddad, I.A.

1987-10-01

The genes coding for apolipoproteins (apo) AI, CIII, and AIV, designated APOA1, APOC3, and APOA4, respectively, are closely linked and tandemly organized in the long arm of the human chromosome 11. A DNA rearrangement involving the genes encoding apoAI and apoCIII in certain patients with premature atherosclerosis has been associated with deficiency of both apoAI and apoCIII in the plasma of these patients. Structural characterization of the genes for apoAI and apoCIII in one of these patients indicates that this rearrangement consists of a DNA inversion containing portions of the 3' ends of the apoAI and apoCIII genes, including themore » DNA region between these genes. The breakpoints of this DNA inversion are located within the fourth exon of the apoAI gene and the first intron of the apoCIII gene. Thus, this DNA inversion results in reciprocal fusion of the apoAI and apoCIII gene transcriptional units. Expression of these gene fusions in cultured mammalian cells results in stable mRNA transcripts with sequences representing fusions of the apoAI and apoCIII mRNAs. These results indicate that absence of transcripts with correct apoAI and apoCIII mRNA sequences causes apoAI and apoCIII deficiency in the plasma of these patients and suggest that these apolipoproteins are involved in cholesterol homeostasis and protection against premature atherosclerosis.« less

A set of highly conserved RNA-binding proteins, alphaCP-1 and alphaCP-2, implicated in mRNA stabilization, are coexpressed from an intronless gene and its intron-containing paralog.

PubMed

Makeyev, A V; Chkheidze, A N; Liebhaber, S A

1999-08-27

Gene families normally expand by segmental genomic duplication and subsequent sequence divergence. Although copies of partially or fully processed mRNA transcripts are occasionally retrotransposed into the genome, they are usually nonfunctional ("processed pseudogenes"). The two major cytoplasmic poly(C)-binding proteins in mammalian cells, alphaCP-1 and alphaCP-2, are implicated in a spectrum of post-transcriptional controls. These proteins are highly similar in structure and are encoded by closely related mRNAs. Based on this close relationship, we were surprised to find that one of these proteins, alphaCP-2, was encoded by a multiexon gene, whereas the second gene, alphaCP-1, was identical to and colinear with its mRNA. The alphaCP-1 and alphaCP-2 genes were shown to be single copy and were mapped to separate chromosomes. The linkage groups encompassing each of the two loci were concordant between mice and humans. These data suggested that the alphaCP-1 gene was generated by retrotransposition of a fully processed alphaCP-2 mRNA and that this event occurred well before the mammalian radiation. The stringent structural conservation of alphaCP-1 and its ubiquitous tissue distribution suggested that the retrotransposed alphaCP-1 gene was rapidly recruited to a function critical to the cell and distinct from that of its alphaCP-2 progenitor.

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

PubMed

Yan, J; Cheng, Q; Li, C B; Aksoy, S

2001-02-01

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

PubMed Central

Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

2010-01-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and the increase of their expression by heat shock and exposure to a hazardous chemical.

PubMed

Iimura, Yosuke; Tatsumi, Kenji

2002-07-01

We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.

Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout.

PubMed

Hidaka, Yoshie; Suzuki, Masakazu

2004-06-01

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

PubMed

Martinez-Nunez, Rocio T; Rupani, Hitasha; Platé, Manuela; Niranjan, Mahesan; Chambers, Rachel C; Howarth, Peter H; Sanchez-Elsner, Tilman

2018-05-16

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction.

PubMed

Papadopoulos, Theofilos; Casemayou, Audrey; Neau, Eric; Breuil, Benjamin; Caubet, Cécile; Calise, Denis; Thornhill, Barbara A; Bachvarova, Magdalena; Belliere, Julie; Chevalier, Robert L; Moulos, Panagiotis; Bachvarov, Dimcho; Buffin-Meyer, Benedicte; Decramer, Stéphane; Auriol, Françoise Conte; Bascands, Jean-Loup; Schanstra, Joost P; Klein, Julie

2017-03-01

Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

PubMed Central

Timpano, Sara; Uniacke, James

2016-01-01

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

eQTL networks unveil enriched mRNA master integrators downstream of complex disease-associated SNPs.

PubMed

Li, Haiquan; Pouladi, Nima; Achour, Ikbel; Gardeux, Vincent; Li, Jianrong; Li, Qike; Zhang, Hao Helen; Martinez, Fernando D; 'Skip' Garcia, Joe G N; Lussier, Yves A

2015-12-01

The causal and interplay mechanisms of Single Nucleotide Polymorphisms (SNPs) associated with complex diseases (complex disease SNPs) investigated in genome-wide association studies (GWAS) at the transcriptional level (mRNA) are poorly understood despite recent advancements such as discoveries reported in the Encyclopedia of DNA Elements (ENCODE) and Genotype-Tissue Expression (GTex). Protein interaction network analyses have successfully improved our understanding of both single gene diseases (Mendelian diseases) and complex diseases. Whether the mRNAs downstream of complex disease genes are central or peripheral in the genetic information flow relating DNA to mRNA remains unclear and may be disease-specific. Using expression Quantitative Trait Loci (eQTL) that provide DNA to mRNA associations and network centrality metrics, we hypothesize that we can unveil the systems properties of information flow between SNPs and the transcriptomes of complex diseases. We compare different conditions such as naïve SNP assignments and stringent linkage disequilibrium (LD) free assignments for transcripts to remove confounders from LD. Additionally, we compare the results from eQTL networks between lymphoblastoid cell lines and liver tissue. Empirical permutation resampling (p<0.001) and theoretic Mann-Whitney U test (p<10(-30)) statistics indicate that mRNAs corresponding to complex disease SNPs via eQTL associations are likely to be regulated by a larger number of SNPs than expected. We name this novel property mRNA hubness in eQTL networks, and further term mRNAs with high hubness as master integrators. mRNA master integrators receive and coordinate the perturbation signals from large numbers of polymorphisms and respond to the personal genetic architecture integratively. This genetic signal integration contrasts with the mechanism underlying some Mendelian diseases, where a genetic polymorphism affecting a single protein hub produces a divergent signal that affects a large number of downstream proteins. Indeed, we verify that this property is independent of the hubness in protein networks for which these mRNAs are transcribed. Our findings provide novel insights into the pleiotropy of mRNAs targeted by complex disease polymorphisms and the architecture of the information flow between the genetic polymorphisms and transcriptomes of complex diseases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

PubMed

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

PubMed

Calliari, Aldo; Farías, Joaquina; Puppo, Agostina; Canclini, Lucía; Mercer, John A; Munroe, David; Sotelo, José R; Sotelo-Silveira, José R

2014-03-01

Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems. Copyright © 2013 Wiley Periodicals, Inc.

Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

PubMed Central

Lapteva, Y. S.; Zolova, O. E.; Shlyapnikov, M. G.; Tsfasman, I. M.; Muranova, T. A.; Stepnaya, O. A.; Kulaev, I. S.

2012-01-01

Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5. PMID:22865082

Cloning and expression analysis of genes encoding lytic endopeptidases L1 and L5 from Lysobacter sp. strain XL1.

PubMed

Lapteva, Y S; Zolova, O E; Shlyapnikov, M G; Tsfasman, I M; Muranova, T A; Stepnaya, O A; Kulaev, I S; Granovsky, I E

2012-10-01

Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

PubMed

Verbeeren, Jens; Verma, Bhupendra; Niemelä, Elina H; Yap, Karen; Makeyev, Eugene V; Frilander, Mikko J

2017-05-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Evidence against translational repression by the carboxyltransferase component of Escherichia coli acetyl coenzyme A carboxylase.

PubMed

Smith, Alexander C; Cronan, John E

2014-11-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evidence against Translational Repression by the Carboxyltransferase Component of Escherichia coli Acetyl Coenzyme A Carboxylase

PubMed Central

Smith, Alexander C.

2014-01-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217–1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. PMID:25157077

mRNA Expression Signatures of Human Skeletal Muscle Atrophy Identify a Natural Compound that Increases Muscle Mass

PubMed Central

Kunkel, Steven D.; Suneja, Manish; Ebert, Scott M.; Bongers, Kale S.; Fox, Daniel K.; Malmberg, Sharon E.; Alipour, Fariborz; Shields, Richard K.; Adams, Christopher M.

2011-01-01

SUMMARY Skeletal muscle atrophy is a common and debilitating condition that lacks a pharmacologic therapy. To develop a potential therapy, we identified 63 mRNAs that were regulated by fasting in both human and mouse muscle, and 29 mRNAs that were regulated by both fasting and spinal cord injury in human muscle. We used these two unbiased mRNA expression signatures of muscle atrophy to query the Connectivity Map, which singled out ursolic acid as a compound whose signature was opposite to those of atrophy-inducing stresses. A natural compound enriched in apples, ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy in mice. It did so by enhancing skeletal muscle insulin/IGF-I signaling, and inhibiting atrophy-associated skeletal muscle mRNA expression. Importantly, ursolic acid’s effects on muscle were accompanied by reductions in adiposity, fasting blood glucose and plasma cholesterol and triglycerides. These findings identify a potential therapy for muscle atrophy and perhaps other metabolic diseases. PMID:21641545

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Last, Jerold A.; Gohil, Kishorchandra; Mathrani, Vivek C.

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkersmore » of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-{kappa}B in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.« less

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

PubMed Central

Sikorav, J L; Duval, N; Anselmet, A; Bon, S; Krejci, E; Legay, C; Osterlund, M; Reimund, B; Massoulié, J

1988-01-01

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms. Images PMID:3181125

RNA-seq analysis of clinical-grade osteochondral allografts reveals activation of early response genes.

PubMed

Lin, Yang; Lewallen, Eric A; Camilleri, Emily T; Bonin, Carolina A; Jones, Dakota L; Dudakovic, Amel; Galeano-Garces, Catalina; Wang, Wei; Karperien, Marcel J; Larson, Annalise N; Dahm, Diane L; Stuart, Michael J; Levy, Bruce A; Smith, Jay; Ryssman, Daniel B; Westendorf, Jennifer J; Im, Hee-Jeong; van Wijnen, Andre J; Riester, Scott M; Krych, Aaron J

2016-11-01

Preservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA-seq) and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assess genome-wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of "early response genes" that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of "early response genes" and reduced levels of ECM-related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1950-1959, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

PubMed Central

Koch, K S; Gleiberman, A S; Aoki, T; Leffert, H L; Feren, A; Jones, A L; Fodor, E J

1995-01-01

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed. Images PMID:7739889

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes.

PubMed

Last, Jerold A; Gohil, Kishorchandra; Mathrani, Vivek C; Kenyon, Nicholas J

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkers of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-kappaB in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.

Metabolic Response of the Cerebral Cortex Following Gentle Sleep Deprivation and Modafinil Administration

PubMed Central

Petit, Jean-Marie; Tobler, Irene; Kopp, Caroline; Morgenthaler, Florence; Borbély, Alexander A.; Magistretti, Pierre J.

2010-01-01

Study Objectives: The main energy reserve of the brain is glycogen, which is almost exclusively localized in astrocytes. We previously reported that cerebral expression of certain genes related to glycogen metabolism changed following instrumental sleep deprivation in mice. Here, we extended our investigations to another set of genes related to glycogen and glucose metabolism. We also compared the effect of instrumentally and pharmacologically induced prolonged wakefulness, followed (or not) by 3 hours of sleep recovery, on the expression of genes related to brain energy metabolism. Design: Sleep deprivation for 6–7 hours. Setting: Animal sleep research laboratory. Participants: Adults OF1 mice. Interventions: Wakefulness was maintained by “gentle sleep deprivation” method (GSD) or by administration of the wakefulness-promoting drug modafinil (MOD) (200 mg/kg i.p.). Measurements and Results: Levels of mRNAs encoding proteins related to energy metabolism were measured by quantitative real-time PCR in the cerebral cortex. The mRNAs encoding protein targeting to glycogen (PTG) and the glial glucose transporter were significantly increased following both procedures used to prolong wakefulness. Glycogenin mRNA levels were increased only after GSD, while neuronal glucose transporter mRNA only after MOD. These effects were reversed after sleep recovery. A significant enhancement of glycogen synthase activity without any changes in glycogen levels was observed in both conditions. Conclusions: These results indicate the existence of a metabolic adaptation of astrocytes aimed at maintaining brain energy homeostasis during the sleep-wake cycle. Citation: Petit, JM; Tobler I; Kopp C; Morgenthaler F; Borbély AA; Magistretti PJ. Metabolic response of the cerebral cortex following gentle sleep deprivation and modafinil administration. SLEEP 2010;33(7):901–908. PMID:20614850

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

PubMed Central

Jackson, Christopher J; Norman, John E; Schnare, Murray N; Gray, Michael W; Keeling, Patrick J; Waller, Ross F

2007-01-01

Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. PMID:17897476

A Yeast Model of FUS/TLS-Dependent Cytotoxicity

PubMed Central

Ju, Shulin; Tardiff, Daniel F.; Han, Haesun; Divya, Kanneganti; Zhong, Quan; Maquat, Lynne E.; Bosco, Daryl A.; Hayward, Lawrence J.; Brown, Robert H.; Lindquist, Susan; Ringe, Dagmar; Petsko, Gregory A.

2011-01-01

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. PMID:21541368

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

PubMed

Swartz, S Zachary; Reich, Adrian M; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M; Campanale, Joseph P; Hamdoun, Amro; Wessel, Gary M

2014-08-01

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. © 2014. Published by The Company of Biologists Ltd.

Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana

PubMed Central

Merret, Rémy; Nagarajan, Vinay K.; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-yung; Green, Pamela J.; Deragon, Jean-Marc; Bousquet-Antonelli, Cécile

2015-01-01

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs. PMID:25845591

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

Reversible methylation of m6Am in the 5′ cap controls mRNA stability

PubMed Central

Mauer, Jan; Luo, Xiaobing; Blanjoie, Alexandre; Jiao, Xinfu; Grozhik, Anya V.; Patil, Deepak P.; Linder, Bastian; Pickering, Brian F.; Vasseur, Jean-Jacques; Chen, Qiuying; Gross, Steven S.; Elemento, Olivier; Debart, Françoise; Kiledjian, Megerditch; Jaffrey, Samie R.

2017-01-01

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. PMID:28002401

mRNA–mRNA duplexes that auto-elicit Staufen1-mediated mRNA decay

PubMed Central

Gong, Chenguang; Tang, Yalan; Maquat, Lynne E.

2013-01-01

We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion. PMID:24056942

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Influenza A Virus Infection of Human Respiratory Cells Induces Primary MicroRNA Expression*

PubMed Central

Buggele, William A.; Johnson, Karen E.; Horvath, Curt M.

2012-01-01

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. In addition to well established means of regulating antiviral gene expression, it has been demonstrated that RNA interference (RNAi) can play an important role in antiviral responses. Virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded microRNA (miRNA) species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates. Examination of miRNA abundance in human lung cell lines revealed endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains, A/Udorn/72 and A/WSN/33. Known antiviral response pathways, including Toll-like receptor, RIG-I-like receptor, and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems. PMID:22822053

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences.

PubMed

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-06-09

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

The human insulin receptor mRNA contains a functional internal ribosome entry segment

PubMed Central

Spriggs, Keith A.; Cobbold, Laura C.; Ridley, Simon H.; Coldwell, Mark; Bottley, Andrew; Bushell, Martin; Willis, Anne E.; Siddle, Kenneth

2009-01-01

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective. PMID:19654240

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences

PubMed Central

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-01-01

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics. PMID:27279482

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p.

PubMed

Cherkasova, Vera; Maury, Luis Lopez; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J

2012-02-01

Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p

PubMed Central

Cherkasova, Vera; Lopez Maury, Luis; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J.

2012-01-01

Deletion of the sla1+ gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1+ have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1–like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1+ (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1+ regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae. PMID:22160596

Epigenetic regulation of lncRNA connects ubiquitin-proteasome system with infection-inflammation in preterm births and preterm premature rupture of membranes.

PubMed

Luo, Xiucui; Pan, Jing; Wang, Leilei; Wang, Peirong; Zhang, Meijiao; Liu, Meilin; Dong, Ziqing; Meng, Qian; Tao, Xuguang; Zhao, Xinliang; Zhong, Julia; Ju, Weina; Gu, Yang; Jenkins, Edmund C; Brown, W Ted; Shi, Qingxi; Zhong, Nanbert

2015-02-15

Preterm premature rupture of membranes (PPROM) is responsible for one third of all preterm births (PTBs). We have recently demonstrated that long noncoding RNAs (lncRNAs) are differentially expressed in human placentas derived from PPROM, PTB, premature rupture of the membranes (PROM), and full-term birth (FTB), and determined the major biological pathways involved in PPROM. Here, we further investigated the relationship of lncRNAs, which are differentially expressed in spontaneous PTB (sPTB) and PPROM placentas and are found to overlap a coding locus, with the differential expression of transcribed mRNAs at the same locus. Ten lncRNAs (five up-regulated and five down-regulated) and the lncRNA-associated 10 mRNAs (six up- and four down-regulated), which were identified by microarray in comparing PPROM vs. sPTB, were then validated by real-time quantitative PCR. A total of 62 (38 up- and 24 down-regulated) and 1,923 (790 up- and 1,133 down-regulated) lncRNAs were identified from placentas of premature labor (sPTB + PPROM), as compared to those from full-term labor (FTB + PROM) and from premature rupture of membranes (PPROM + PROM), as compared to those from non-rupture of membranes (sPTB + FTB), respectively. We found that a correlation existed between differentially expressed lncRNAs and their associated mRNAs, which could be grouped into four categories based on the gene strand (sense or antisense) of lncRNA and its paired transcript. These findings suggest that lncRNA regulates mRNA transcription through differential mechanisms. Differential expression of the transcripts PPP2R5C, STAM, TACC2, EML4, PAM, PDE4B, STAM, PPP2R5C, PDE4B, and EGFR indicated a co-expression among these mRNAs, which are involved in the ubiquitine-proteasome system (UPS), in addition to signaling transduction and beta adrenergic signaling, suggesting that imbalanced regulation of UPS may present an additional mechanism underlying the premature rupture of membrane in PPROM. Differentially expressed lncRNAs that were identified from the human placentas of sPTB and PPROM may regulate their associated mRNAs through differential mechanisms and connect the ubiquitin-proteasome system with infection-inflammation pathways. Although the detailed mechanisms by which lncRNAs regulate their associated mRNAs in sPTB and PPROM are yet to be clarified, our findings open a new approach to explore the pathogenesis of sPTB and PPROM.

Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression

PubMed Central

Yee, Janet; Tang, Anita; Lau, Wei-Ling; Ritter, Heather; Delport, Dewald; Page, Melissa; Adam, Rodney D; Müller, Miklós; Wu, Gang

2007-01-01

Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him) is essential for its activity. The Giardia core histone genes are constitutively expressed at approximately equivalent levels and their mRNAs are polyadenylated. Competition gel-shift experiments suggest that a factor within the protein complex that binds him may also be a part of the protein complexes that bind other promoter elements described previously in Giardia. Conclusion In contrast to other eukaryotes, the Giardia genome has only a single class of core histone genes that encode replication-independent histones. Our inability to locate a gene encoding the linker histone H1 leads us to speculate that the H1 protein may not be required for the compaction of Giardia's small and gene-rich genome. PMID:17425802

Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection

PubMed Central

Mehdy, Mona C.; Lamb, Christopher J.

1987-01-01

The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A λgt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 105 recombinants with an antiserum to purified bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. Addition of elicitor to cell cultures resulted in the rapid accumulation of CHI mRNA, with maximum levels achieved 3–4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals were strikingly similar to those of mRNAs encoding two other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families encoding these two enzymes, chalcone isomerase is encoded by a single gene which is regulated by several environmental stimuli. ImagesFig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 9. PMID:16453768

Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium.

PubMed

Whitaker, L H R; Murray, A A; Matthews, R; Shaw, G; Williams, A R W; Saunders, P T K; Critchley, H O D

2017-03-01

What is the impact of administration of the selective progesterone receptor modulator (SPRM), ulipristal acetate (UPA) on the endometrium of women with fibroids? UPA administration altered expression of sex-steroid receptors and progesterone-regulated genes and was associated with low levels of glandular and stromal cell proliferation. Administration of all SPRM class members results in PAEC (progesterone receptor modulator associated endometrial changes). Data on the impact of the SPRM UPA administration on endometrial sex-steroid receptor expression, progesterone (P)-regulated genes and cell proliferation are currently lacking. Observational study with histological and molecular analyses to delineate impact of treatment with UPA on endometrium. Endometrial samples (n = 9) were collected at hysterectomy from women aged 39 to 49 with uterine fibroids treated with UPA (oral 5 mg daily) for 9-12 weeks. Control proliferative (n = 9) and secretory (n = 9) endometrium from women aged 38-52 with fibroids were derived from institutional tissue archives. Study setting was a University Research Institute. Endometrial biopsies were collected with institutional ethical approval and written informed consent. Concentrations of mRNAs encoded by steroid receptors, P-regulated genes and factors in decidualised endometrium were quantified with qRT-PCR. Immunohistochemistry was employed for localization of progesterone (PR, PRB), androgen (AR), estrogen (ERα) receptors and expression of FOXO1, HAND2, HOXA10, PTEN homologue. Endometrial glandular and stromal cell proliferation was objectively quantified using Ki67. UPA induced morphological changes in endometrial tissue consistent with PAEC. A striking change in expression patterns of PR and AR was detected compared with either proliferative or secretory phase samples. There were significant changes in pattern of expression of mRNAs encoded by IGFBP-1, FOXO1, IL-15, HAND2, IHH and HOXA10 compared with secretory phase samples consistent with low agonist activity in endometrium. Expression of mRNA encoded by FOXM1, a transcription factor implicated in cell cycle progression, was low in UPA-treated samples. Cell proliferation (Ki67 positive nuclei) was lower in samples from women treated with UPA compared with those in the proliferative phase. N/A. A small number of well-characterized patients were studied in-depth. The impacts on morphology, molecular and cellular changes with SPRM, UPA administration on symptom control remains to be determined. P plays a pivotal role in endometrial function. P-action is mediated through interaction with the PR. These data provide support for onward development of the SPRM class of compounds as effective long-term medical therapy for heavy menstrual bleeding. H.O.D.C. received has clinical research support for laboratory consumables and staff from Bayer Pharma Ag and provides consultancy advice (no personal remuneration) for Bayer Pharma Ag, PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc.; A.R.W.W. has received consultancy payments from Bayer, Gedeon Richter, Preglem SA, HRA Pharma; L.H.R.W., A.A.M., R.M., G.S. and P.T.K.S. have no conflicts of interest. Study funded in part from each of: Medical Research Council (G1002033; G1100356/1; MR/N022556/1); National Health Institute for Health Research (12/206/520) and TENOVUS Scotland. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

T Cell Post-Transcriptional miRNA-mRNA Interaction Networks Identify Targets Associated with Susceptibility/Resistance to Collagen-induced Arthritis

PubMed Central

Macedo, Claudia; Cunha, Thiago M.; Nascimento, Daniele C. B.; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Cunha, Fernando Q.; Passos, Geraldo A.

2013-01-01

Background Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. Methodology/Principal Findings To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3+ T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir++ algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3′UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. Conclusions/Significance This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA. PMID:23359619

Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed Central

Floriano, B; Herrero, A; Flores, E

1994-01-01

A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures. Images PMID:7929012

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

PubMed

Kishi, Masashi; Kummer, Terrance T; Eglen, Stephen J; Sanes, Joshua R

2005-04-25

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

PubMed

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

PubMed Central

Corcoran, Jennifer A.; Khaperskyy, Denys A.; Johnston, Benjamin P.; King, Christine A.; Cyr, David P.; Olsthoorn, Alisha V.

2012-01-01

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells. PMID:22696654

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

PubMed Central

Verbeeren, Jens; Verma, Bhupendra

2017-01-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3′ untranslated region (3′UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3′UTR to non-productive isoforms with a long 3′UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5′ splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3′UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm. PMID:28549066

Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities*

PubMed Central

Schütt, Janin; Falley, Katrin; Richter, Dietmar; Kreienkamp, Hans-Jürgen; Kindler, Stefan

2009-01-01

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients. PMID:19640847

The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing the translation of regulatory and structural genes.

PubMed

Moreno, Renata; Fonseca, Pilar; Rojo, Fernando

2010-08-06

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

PubMed

Hernández-Arranz, Sofía; Moreno, Renata; Rojo, Fernando

2013-01-01

Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

PubMed

Ericsson, A; Geenen, V; Robert, F; Legros, J J; Vrindts-Gevaert, Y; Franchimont, P; Brene, S; Persson, H

1990-08-01

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.

Multi-step splicing of sphingomyelin synthase linear and circular RNAs.

PubMed

Filippenkov, Ivan B; Sudarkina, Olga Yu; Limborska, Svetlana A; Dergunova, Lyudmila V

2018-05-15

The SGMS1 gene encodes the enzyme sphingomyelin synthase 1 (SMS1), which is involved in the regulation of lipid metabolism, apoptosis, intracellular vesicular transport and other significant processes. The SGMS1 gene is located on chromosome 10 and has a size of 320 kb. Previously, we showed that dozens of alternative transcripts of the SGMS1 gene are present in various human tissues. In addition to mRNAs that provide synthesis of the SMS1 protein, this gene participates in the synthesis of non-coding transcripts, including circular RNAs (circRNAs), which include exons of the 5'-untranslated region (5'-UTR) and are highly represented in the brain. In this study, using the high-throughput technology RNA-CaptureSeq, many new SGMS1 transcripts were identified, including both intronic unspliced RNAs (premature RNAs) and RNAs formed via alternative splicing. Recursive exons (RS-exons) that can participate in the multi-step splicing of long introns of the gene were also identified. These exons participate in the formation of circRNAs. Thus, multi-step splicing may provide a variety of linear and circular RNAs of eukaryotic genes in tissues. Copyright © 2018 Elsevier B.V. All rights reserved.

IDPT: Insights into potential intrinsically disordered proteins through transcriptomic analysis of genes for prostate carcinoma epigenetic data.

PubMed

Mallik, Saurav; Sen, Sagnik; Maulik, Ujjwal

2016-07-15

Involvement of intrinsically disordered proteins (IDPs) with various dreadful diseases like cancer is an interesting research topic. In order to gain novel insights into the regulation of IDPs, in this article, we perform a transcriptomic analysis of mRNAs (genes) for transcripts encoding IDPs on a human multi-omics prostate carcinoma dataset having both gene expression and methylation data. In this regard, firstly the genes that consist of both the expression and methylation data, and that are corresponding to the cancer-related prostate-tissue-specific disordered proteins of MobiDb database, are selected. We apply standard t-test for determining differentially expressed genes as well as differentially methylated genes. A network having these genes and their targeter miRNAs from Diana Tarbase v7.0 database and corresponding Transcription Factors from TRANSFAC and ITFP databases, is then built. Thereafter, we perform literature search, and KEGG pathway and Gene Ontology analyses using DAVID database. Finally, we report several significant potential gene-markers (with the corresponding IDPs) that have inverse relationship between differential expression and methylation patterns, and that are hub genes of the TF-miRNA-gene network. Copyright © 2016 Elsevier B.V. All rights reserved.

EYA1 mutations associated with the branchio-oto-renal syndrome result in defective otic development in Xenopus laevis

PubMed Central

Li, Youe; Manaligod, Jose M.; Weeks, Daniel L.

2009-01-01

Background information. The BOR (branchio-oto-renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. Results. We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr-1 and nt3) important for normal ear development. Conclusions. These studies lend support to the hypothesis that dominant-negative effects of EYA1 mutations may have a role in the pathogenesis of BOR. PMID:19951260

Antisense apolipoprotein B therapy: where do we stand?

PubMed

Akdim, Fatima; Stroes, Erik S G; Kastelein, John J P

2007-08-01

Antisense oligonucleotides are novel therapeutic agents that reduce the number of specific mRNAs available for translation of the encoded protein. ISIS 301012 is an antisense oligonucleotide developed to reduce the hepatic synthesis of apolipoprotein B-100. Apolipoprotein B-100 is made in the liver, and antisense oligonucleotides preferentially distribute to that organ, so antisense apolipoprotein B-100 may have potential as an efficacious lipid-lowering agent. Recently, in healthy volunteers and in mild dyslipidaemic patients, this strategy as monotherapy or in conjunction with statins has shown unparalleled efficacy in reducing apolipoprotein B-100 and LDL-cholesterol. Tolerance for this novel therapy is encouraging and safety concerns currently only relate to mild injection-site reactions and rare liver-function test abnormalities. It should be noted, however, that these safety results were obtained in relatively few individuals. ISIS 301012 has initially shown promising results in experimental animal models, and in clinical trials in humans. Besides the effect of reducing apolipoprotein B-100 and LDL-cholesterol, this compound also significantly lowers plasma triglycerides. Safety concerns related to the drug include increased liver-function tests. To date no evidence of hepatic steatosis has been reported. Nonetheless, clinical trials of longer duration are required to demonstrate further safety.

Messenger RNA for membrane-type 2 matrix metalloproteinase, MT2-MMP, is expressed in human placenta of first trimester.

PubMed

Bjørn, S F; Hastrup, N; Larsen, J F; Lund, L R; Pyke, C

2000-01-01

An intimately regulated cell surface activation of matrix metalloproteinases (MMPs) is believed to be of critical importance for the control of trophoblast invasion. A histological investigation of the expression and localization of three different MMPs, the membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP, MT2-MMP) and matrix metalloproteinase 2 (MMP-2/gelatinase A) was performed by in situ hybridization on consecutive sections from human placentae of first trimester pregnancies. Cytokeratin immunostaining identified trophoblast cells. Both normal and tubal implantation sites were studied. We observed a high degree of coexpression of MT2-MMP, MT1-MMP and MMP-2 mRNAs in single extravillous cytotrophoblasts that had invaded the endometrium and tubal wall. Furthermore, mRNAs for all three genes were also seen in cytotrophoblasts of cell islands. In contrast to this coexpression pattern, MT2-MMP expression was absent from cell columns and decidual cells, in which signals for MT1-MMP and MMP-2 mRNAs were seen. The present data on the cellular expression of MT2-MMP mRNA in placenta extend our knowledge of the proteolytic events that take place during early pregnancy. The data suggest that MT2-MMP, capable of activating MMP-2 in vitro, is involved in the invasion of extravillous cytotrophoblast, possibly related to the physiological activation of MMP-2. Copyright 2000 Harcourt Publishers Ltd.

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

PubMed

Gonzalez, Gaëlle; Vituret, Cyrielle; Di Pietro, Attilio; Chanson, Marc; Boulanger, Pierre; Hong, Saw-See

2012-01-01

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes

PubMed Central

Occhipinti, Laura; Kemmler, Stefan; Panse, Vikram G.

2012-01-01

Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation. PMID:22956913

mRNAs for PRPs, statherin, and histatins in von Ebner's gland tissues.

PubMed

Azen, E A; Hellekant, G; Sabatini, L M; Warner, T F

1990-11-01

A search was made for expression of genes for proline-rich proteins (PRPs) and other salivary-type proteins, including statherin and histatins, in taste-bud tissues of mice and primates because of previous genetic findings in mice (Azen et al., 1986) that Prp and taste genes for certain bitter substances are either the same or closely linked. Taste-bud tissues and other tissues were tested for specific mRNAs with labeled DNA probes by Northern blotting and in situ hybridization. It was found that PRP mRNAs were present in von Ebner's glands of mice and macaques, and that there was a much greater degree of PRP mRNA induction in mouse parotid (16-fold) than in von Ebner's gland (two-fold) after in vivo isoproterenol stimulation. This difference may be due, in part, to differences in autonomic nerve innervation. Statherin and histatin mRNAs were found in macaque taste-bud tissues containing von Ebner's gland, and statherin protein was found in human von Ebner's gland by immunohistochemistry. The finding of PRP gene expression in von Ebner's gland, whose secretions have been suggested to play a role in taste stimulation, adds further support to a possible function of PRPs in bitter tasting. The possible functions of statherin and histatins in von Ebner's gland secretions may be related to statherin's regulation of salivary calcium and histatins' antibacterial and antifungal properties.

The Role of Telomeric Repeat Binding Factor 1 (TRF1) in Telomere Maintenance and as a Potential Prognostic Indicator in Human Breast Cancer

DTIC Science & Technology

2006-04-01

for Specific Aim #3 have yet been initiated, and are proceeding on schedule. The PhD candidate has completed her educational goals. 13 Appendix A ... LEVELS OF TELOMERE PROTEIN MRNAS ARE PREDICTIVE OF TELOMERE CONTENT IN HUMAN BREAST TUMORS Kimberly S. Butler, William C. Hines, Diana Roberts

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome

PubMed Central

Sakurai, Masayuki; Ueda, Hiroki; Yano, Takanori; Okada, Shunpei; Terajima, Hideki; Mitsuyama, Toutai; Toyoda, Atsushi; Fujiyama, Asao; Kawabata, Hitomi; Suzuki, Tsutomu

2014-01-01

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called “inosine chemical erasing” (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions. PMID:24407955

MiR-21 is enriched in the RNA-induced silencing complex and targets COL4A1 in human granulosa cell lines.

PubMed

Mase, Yuri; Ishibashi, Osamu; Ishikawa, Tomoko; Takizawa, Takami; Kiguchi, Kazushige; Ohba, Takashi; Katabuchi, Hidetaka; Takeshita, Toshiyuki; Takizawa, Toshihiro

2012-10-01

MicroRNAs (miRNAs) are noncoding small RNAs that play important roles in a variety of physiological and pathological events. In this study, we performed large-scale profiling of EIF2C2-bound miRNAs in 3 human granulosa-derived cell lines (ie, KGN, HSOGT, and GC1a) by high-throughput sequencing and found that miR-21 accounted for more than 80% of EIF2C2-bound miRNAs, suggesting that it was enriched in the RNA-induced silencing complex (RISC) and played a functional role in human granulosa cell (GC) lines. We also found high expression levels of miR-21 in primary human GCs. Assuming that miR-21 target mRNAs are enriched in RISC, we performed cDNA cloning of EIF2C2-bound mRNAs in KGN cells. We identified COL4A1 mRNA as a miR-21 target in the GC lines. These data suggest that miR-21 is involved in the regulation of the synthesis of COL4A1, a component of the basement membrane surrounding the GC layer and granulosa-embedded extracellular structure.

Multiple Export Mechanisms for mRNAs

PubMed Central

Delaleau, Mildred; Borden, Katherine L. B.

2015-01-01

Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay

PubMed Central

Baird, Thomas D; Cheng, Ken Chih-Chien; Chen, Yu-Chi; Buehler, Eugen; Martin, Scott E; Inglese, James

2018-01-01

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5–10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC. PMID:29528287

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

HomoTarget: a new algorithm for prediction of microRNA targets in Homo sapiens.

PubMed

Ahmadi, Hamed; Ahmadi, Ali; Azimzadeh-Jamalkandi, Sadegh; Shoorehdeli, Mahdi Aliyari; Salehzadeh-Yazdi, Ali; Bidkhori, Gholamreza; Masoudi-Nejad, Ali

2013-02-01

MiRNAs play an essential role in the networks of gene regulation by inhibiting the translation of target mRNAs. Several computational approaches have been proposed for the prediction of miRNA target-genes. Reports reveal a large fraction of under-predicted or falsely predicted target genes. Thus, there is an imperative need to develop a computational method by which the target mRNAs of existing miRNAs can be correctly identified. In this study, combined pattern recognition neural network (PRNN) and principle component analysis (PCA) architecture has been proposed in order to model the complicated relationship between miRNAs and their target mRNAs in humans. The results of several types of intelligent classifiers and our proposed model were compared, showing that our algorithm outperformed them with higher sensitivity and specificity. Using the recent release of the mirBase database to find potential targets of miRNAs, this model incorporated twelve structural, thermodynamic and positional features of miRNA:mRNA binding sites to select target candidates. Copyright © 2012 Elsevier Inc. All rights reserved.

SMN is essential for the biogenesis of U7 snRNP and 3′-end formation of histone mRNAs

PubMed Central

Tisdale, Sarah; Lotti, Francesco; Saieva, Luciano; Van Meerbeke, James P.; Crawford, Thomas O.; Sumner, Charlotte J.; Mentis, George Z.; Pellizzoni, Livio

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the survival motor neuron (SMN) protein. SMN mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs. Here we investigated SMN requirement for the biogenesis and function of U7—an snRNP specialized in the 3′-end formation of replication-dependent histone mRNAs that normally are not polyadenylated. We show that SMN deficiency impairs U7 snRNP assembly and decreases U7 levels in mammalian cells. The SMN-dependent U7 reduction affects endonucleolytic cleavage of histone mRNAs leading to abnormal accumulation of 3′-extended and polyadenylated transcripts, followed by downstream changes in histone gene expression. Importantly, SMN deficiency induces defects of histone mRNA 3′-end formation in both SMA mice and human patients. These findings demonstrate that SMN is essential for U7 biogenesis and histone mRNA processing in vivo, and identify a novel RNA pathway disrupted in SMA. PMID:24332368

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

PubMed Central

Vanderperre, Benoît; Lucier, Jean-François; Bissonnette, Cyntia; Motard, Julie; Tremblay, Guillaume; Vanderperre, Solène; Wisztorski, Maxence; Salzet, Michel; Boisvert, François-Michel; Roucou, Xavier

2013-01-01

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome. PMID:23950983

Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans

PubMed Central

van Boheemen, Sander; de Graaf, Miranda; Lauber, Chris; Bestebroer, Theo M.; Raj, V. Stalin; Zaki, Ali Moh; Osterhaus, Albert D. M. E.; Haagmans, Bart L.; Gorbalenya, Alexander E.; Snijder, Eric J.; Fouchier, Ron A. M.

2012-01-01

ABSTRACT A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genome was reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C. PMID:23170002

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis.

PubMed

Drummond, Micah J; Miyazaki, Mitsunori; Dreyer, Hans C; Pennings, Bart; Dhanani, Shaheen; Volpi, Elena; Esser, Karyn A; Rasmussen, Blake B

2009-04-01

Muscle growth is associated with an activation of the mTOR signaling pathway and satellite cell regulators. The purpose of this study was to determine whether 17 selected genes associated with mTOR/muscle protein synthesis and the satellite cells/myogenic program are differentially expressed in young and older human skeletal muscle at rest and in response to a potent anabolic stimulus [resistance exercise + essential amino acid ingestion (RE+EAA)]. Twelve male subjects (6 young, 6 old) completed a bout of heavy resistance exercise. Muscle biopsies were obtained before and at 3 and 6 h post RE+EAA. Subjects ingested leucine-enriched essential amino acids at 1 h postexercise. mRNA expression was determined using qRT-PCR. At rest, hVps34 mRNA was elevated in the older subjects (P < 0.05) while there was a tendency for levels of myoD, myogenin, and TSC2 mRNA to be higher than young. The anabolic stimulus (RE+EAA) altered mRNAs associated with mTOR regulation. Notably, REDD2 decreased in both age groups (P < 0.05) but the expression of Rheb mRNA increased only in the young. Finally, cMyc mRNA was elevated (P < 0.05) in both young and old at 6 h post RE+EAA. Furthermore, RE+EAA also increased expression of several mRNAs associated with satellite function in the young (P < 0.05), while expression of these mRNAs did not change in the old. We conclude that several anabolic genes in muscle are more responsive in young men post RE+EAA. Our data provide new insights into the regulation of genes important for transcription and translation in young and old human skeletal muscle post RE+EAA.

Effects of spaceflight on hypothalamic peptide systems controlling pituitary growth hormone dynamics

NASA Technical Reports Server (NTRS)

Sawchenko, P. E.; Arias, C.; Krasnov, I.; Grindeland, R. E.; Vale, W.

1992-01-01

Possible effects of reduced gravity on central hypophysiotropic systems controlling growth hormone (GH) secretion were investigated in rats flown on Cosmos 1887 and 2044 biosatellites. Immunohistochemical (IHC)staining for the growth hormone-releasing factor (GRF), somatostatin (SS), and other hypothalamic hormones was performed on hypothalami obtained from rats. IHC analysis was complemented by quantitative in situ assessments of mRNAs encoding the precursors for these hormones. Data obtained suggest that exposure to microgravity causes a preferential reduction in GRF peptide and mRNA levels in hypophysiotropic neurons, which may contribute to impared GH secretion in animals subjected to spaceflight. Effects of weightlessness are not mimicked by hindlimb suspension in this system.

Mechanisms and consequences of alternative polyadenylation

PubMed Central

Di Giammartino, Dafne Campigli; Nishida, Kensei; Manley, James L.

2011-01-01

Summary Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3’UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechansims governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenlyation. PMID:21925375

Estrogen Receptor Mutants/Variants in Human Breast Cancer.

DTIC Science & Technology

1996-12-01

average 1 hour per response, including the time for reviewing instructions, searching existing data sources,gathering and maintaining the data needed...Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188...we identified, for the first time , the expression of exon deleted progesterone receptor (PR) mRNAs in both normal and neoplastic human breast tissues

The Unicellular Green Alga Chlamydomonas reinhardtii as an Experimental System to Study Chloroplast RNA Metabolism

NASA Astrophysics Data System (ADS)

Nickelsen, J.; Kück, U.

Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

Spatial epigenetics: linking nuclear structure and function in higher eukaryotes.

PubMed

Jackson, Dean A

2010-09-20

Eukaryotic cells are defined by the genetic information that is stored in their DNA. To function, this genetic information must be decoded. In doing this, the information encoded in DNA is copied first into RNA, during RNA transcription. Primary RNA transcripts are generated within transcription factories, where they are also processed into mature mRNAs, which then pass to the cytoplasm. In the cytoplasm these mRNAs can finally be translated into protein in order to express the genetic information as a functional product. With only rare exceptions, the cells of an individual multicellular eukaryote contain identical genetic information. However, as different genes must be expressed in different cell types to define the structure and function of individual tissues, it is clear that mechanisms must have evolved to regulate gene expression. In higher eukaryotes, mechanisms that regulate the interaction of DNA with the sites where nuclear functions are performed provide one such layer of regulation. In this chapter, I evaluate how a detailed understanding of nuclear structure and chromatin dynamics are beginning to reveal how spatial mechanisms link chromatin structure and function. As these mechanisms operate to modulate the genetic information in DNA, the regulation of chromatin function by nuclear architecture defines the concept of 'spatial epigenetics'.

Loss of function of a rice TPR-domain RNA-binding protein confers broad-spectrum disease resistance.

PubMed

Zhou, Xiaogang; Liao, Haicheng; Chern, Mawsheng; Yin, Junjie; Chen, Yufei; Wang, Jianping; Zhu, Xiaobo; Chen, Zhixiong; Yuan, Can; Zhao, Wen; Wang, Jing; Li, Weitao; He, Min; Ma, Bingtian; Wang, Jichun; Qin, Peng; Chen, Weilan; Wang, Yuping; Liu, Jiali; Qian, Yangwen; Wang, Wenming; Wu, Xianjun; Li, Ping; Zhu, Lihuang; Li, Shigui; Ronald, Pamela C; Chen, Xuewei

2018-03-20

Crops carrying broad-spectrum resistance loci provide an effective strategy for controlling infectious disease because these loci typically confer resistance to diverse races of a pathogen or even multiple species of pathogens. Despite their importance, only a few crop broad-spectrum resistance loci have been reported. Here, we report the identification and characterization of the rice bsr-k1 (broad-spectrum resistance Kitaake-1) mutant, which confers broad-spectrum resistance against Magnaporthe oryzae and Xanthomonas oryzae pv oryzae with no major penalty on key agronomic traits. Map-based cloning reveals that Bsr-k1 encodes a tetratricopeptide repeats (TPRs)-containing protein, which binds to mRNAs of multiple OsPAL ( OsPAL1-7 ) genes and promotes their turnover. Loss of function of the Bsr-k1 gene leads to accumulation of OsPAL1-7 mRNAs in the bsr-k1 mutant. Furthermore, overexpression of OsPAL1 in wild-type rice TP309 confers resistance to M. oryzae , supporting the role of OsPAL1 Our discovery of the bsr-k1 allele constitutes a significant conceptual advancement and provides a valuable tool for breeding broad-spectrum resistant rice. Copyright © 2018 the Author(s). Published by PNAS.

Role of ATF4 in skeletal muscle atrophy.

PubMed

Adams, Christopher M; Ebert, Scott M; Dyle, Michael C

2017-05-01

Here, we discuss recent work focused on the role of activating transcription factor 4 (ATF4) in skeletal muscle atrophy. Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression; however, the transcriptional regulatory proteins responsible for those changes are not yet well defined. Recent work indicates that some forms of muscle atrophy require ATF4, a stress-inducible bZIP transcription factor subunit that helps to mediate a broad range of stress responses in mammalian cells. ATF4 expression in skeletal muscle fibers is sufficient to induce muscle fiber atrophy and required for muscle atrophy during several stress conditions, including aging, fasting, and limb immobilization. By helping to activate specific genes in muscle fibers, ATF4 contributes to the expression of numerous mRNAs, including at least two mRNAs (Gadd45a and p21) that encode mediators of muscle fiber atrophy. Gadd45a promotes muscle fiber atrophy by activating the protein kinase MEKK4. p21 promotes atrophy by reducing expression of spermine oxidase, a metabolic enzyme that helps to maintain muscle fiber size under nonstressed conditions. In skeletal muscle fibers, ATF4 is critical component of a complex and incompletely understood molecular signaling network that causes muscle atrophy during aging, fasting, and immobilization.

SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation

PubMed Central

Matsuda, Morihiro; Korn, Bobby S.; Hammer, Robert E.; Moon, Young-Ah; Komuro, Ryutaro; Horton, Jay D.; Goldstein, Joseph L.; Brown, Michael S.; Shimomura, Iichiro

2001-01-01

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting–refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions. PMID:11358865

Retrieval of Missing Spliced Leader in Dinoflagellates

PubMed Central

Zhang, Huan; Lin, Senjie

2009-01-01

Spliced leader (SL) trans-splicing has recently been shown to be a common mRNA processing mechanism in dinoflagellates, in which a short (22-nt) sequence, DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), is transplanted from the 5′-end of a small non-coding RNA (SL RNA) to the 5′ end of mRNA molecules. The widespread existence of the mechanism in dinoflagellates has been demonstrated by detection of this SL (DinoSL) in a wide phylogenetic range of dinoflagellates. Furthermore, the presence of DinoSL in the transcripts of highly diverse groups of nuclear-encoded genes has led us to postulate that SL trans-splicing is universal in dinoflagellate nuclear genome. However, some observations inconsistent to this postulation have been reported, exemplified by a recent article reporting apparent absence of DinoSL in the transcripts of some nuclear-encoded genes in Amphidinium carterae. Absence of SL in these gene transcripts would have important implication on gene regulation in dinoflagellates and utility of DinoSL as a universal dinoflagellate-specific primer to study dinoflagellate transcriptomics. In this study, we re-examined transcripts of these genes and found that all of them actually contained DinoSL. Therefore, results to date are consistent to our initial postulation that DinoSL occurs in all dinoflagellate nuclear-encoded mRNAs. PMID:19122814

[Variational structure and function of products from IGF-1 gene].

PubMed

Zhang, Bing-Bing; Wang, Yuan-Liang; Fan, Kai

2008-07-01

The IGF-1 gene, containing six exons, is characterized by the generation of multiple heterogeneous mRNA transcripts and translations. The IGF-1 isoforms being produced arise from the combination of multiple transcription initiation sites, alternate splicing, and different polyadenylation signals. These different mRNAs are translated to distinct circulating and local isoforms. The circulating mature IGF-1 is encoded by exons 3 and 4, and its biological function in growth and development has been intensively studied. The local isoforms of IGF-1 contains the part encoded by exons 3 and 4, and moreover the alternate extension peptide at carboxy-terminal, encoded by exons 5 and 6, is also included in the isoforms. And the functions of local IGF-1 isoforms and E-peptides have been overlooked until recently. Recently investigation shows that cell discrepant response to the overexpression of different IGF-1 isoforms and the E-peptides, and more interestingly, IGF-1Ea, IGF-1Eb (MGF) and MGF E-peptide have potential to promote skeletal muscle regeneration, to prevent cardiac muscle loss and neural damage. The acting mechanism of IGF-1 isoforms differ from the IGF-1, and the isoforms functioned probably by binding to specific E-peptide receptor, instead of binding to the IGF-1R.

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction 1

PubMed Central

Vernon, Daniel M.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C3 photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte. ImagesFigure 1Figure 2 PMID:16669095

Sexual selection, genetic conflict, selfish genes, and the atypical patterns of gene expression in spermatogenic cells.

PubMed

Kleene, Kenneth C

2005-01-01

This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.

EsrE-A yigP Locus-Encoded Transcript-Is a 3′ UTR sRNA Involved in the Respiratory Chain of E. coli

PubMed Central

Xia, Hui; Yang, Xichen; Tang, Qiongwei; Ye, Jiang; Wu, Haizhen; Zhang, Huizhan

2017-01-01

The yigP locus is widely conserved among γ-proteobacteria. Mutation of the yigP locus impacts aerobic growth of Gram-negative bacteria. However, the underlying mechanism of how the yigP locus influences aerobic growth remains largely unknown. Here, we demonstrated that the yigP locus in Escherichia coli encodes two transcripts; the mRNA of ubiquinone biosynthesis protein, UbiJ, and the 3′ untranslated region small regulatory RNA (sRNA), EsrE. EsrE is an independent transcript that is transcribed using an internal promoter of the yigP locus. Surprisingly, we found that both the EsrE sRNA and UbiJ protein were required for Q8 biosynthesis, and were sufficient to rescue the growth defect ascribed to deletion of the yigP locus. Moreover, our data showed that EsrE targeted multiple mRNAs involved in several cellular processes including murein biosynthesis and the tricarboxylic acid cycle. Among these targets, sdhD mRNA that encodes one subunit of succinate dehydrogenase (SDH), was significantly activated. Our findings provided an insight into the important function of EsrE in bacterial adaptation to various environments, as well as coordinating different aspects of bacterial physiology. PMID:28900423

Gestational form of Selenium in Free-Choice Mineral Mixes Affects Transcriptome Profiles of the Neonatal Calf Testis, Including those of Steroidogenic and Spermatogenic Pathways.

PubMed

Cerny, K L; Garbacik, S; Skees, C; Burris, W R; Matthews, J C; Bridges, P J

2016-01-01

In areas where soils are deficient in Selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Because Se status affects testosterone synthesis and frequency of sperm abnormalities, and the form of Se supplemented to cows affects tissue-specific gene expression, the objective of this study was to determine whether the form of Se consumed by cows during gestation would affect the expression of mRNAs that regulate steroidogenesis and/or spermatogenesis in the neonatal calf testis. Twenty-four predominantly Angus cows were assigned randomly to have individual, ad libitum, access of a mineral mix containing 35 ppm of Se in free-choice vitamin-mineral mixes as either inorganic (ISe), organic (OSe), or a 50/50 mix of ISe and OSe (MIX), starting 4 months prior to breeding and continuing throughout gestation. Thirteen male calves were born over a 3-month period (ISe, n = 5; OSe, n = 4; MIX, n = 4), castrated within 2 days of birth, and extracted testis RNA subjected to transcriptomal analysis by microarray (Affymetrix Bovine 1.0 ST arrays) and targeted gene expression analysis by real-time reverse-transcription PCR (RT-PCR) of mRNAs encoding proteins known to affect steroidogenesis and/or spermatogenesis. The form of dam Se affected (P < 0.05) the expression of 853 annotated genes, including 17 mRNAs putatively regulating steroidogenesis and/or spermatogenesis. Targeted RT-PCR analysis indicated that the expression of mRNA encoding proteins CYP2S1 (cytochrome P450, family 2, subfamily S, polypeptide 1), HSD17B7 (hydroxysteroid (17β) dehydrogenase 7), SULT1E1 (sulfotransferase family 1E, estrogen preferring, member 1), LDHA (lactate dehydrogenase A), CDK5R1 (cyclin-dependent kinase 5, regulatory subunit 1), and LEP (leptin) was affected (P < 0.05) by form of Se consumed by dams of developing bull calves, while AKR1C4 (aldo-keto reductase family 1, member C4) and CCND2 (cyclin D2) tended (P < 0.09) to be affected. Our results indicate that form of Se fed to dams during gestation affected the transcriptome of the neonatal calf testis. If these profiles are maintained throughout maturation, then the form of Se fed to dams may impact bull fertility and the development of Se form-dependent mineral mixes that target gestational development of the testis are warranted.

Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

PubMed

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

2009-09-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

Existence of a microRNA pathway in anucleate platelets

PubMed Central

Landry, Patricia; Plante, Isabelle; Ouellet, Dominique L; Perron, Marjorie P; Rousseau, Guy; Provost, Patrick

2010-01-01

Platelets play a critical role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlie stroke and acute coronary syndromes. Anucleate platelets contain messenger RNAs (mRNAs) and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation. Further analyses revealed that platelets contain Dicer and Argonaute 2 (Ago2) complexes functional in exogenously supplied miRNA precursor (pre-miRNA) processing and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y12 mRNA in Ago2 immunoprecipitates suggests that P2Y12 expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. PMID:19668211

Dual Functions of Rift Valley Fever Virus NSs Protein: Inhibition of Host mRNA Transcription and Post-transcriptional Downregulation of Protein Kinase PKR

PubMed Central

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C. J.; Makino, Shinji

2011-01-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-β mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase, PKR, to prevent phosphorylation of eIF2α and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts. PMID:19751406

LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

PubMed

Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

2017-11-28

Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm

PubMed Central

Sundarrajan, Lakshminarasimhan; Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Canosa, Luis Fabián; Unniappan, Suraj

2016-01-01

Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT- qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1. PMID:27329836

Can glomerular mRNAs in human type 1 diabetes be used to predict transition from normoalbuminuria to microalbuminuria?

PubMed

Adler, Sharon G; Kang, Shin-Wook; Feld, Stella; Cha, Dae Ryong; Barba, Lilly; Striker, Liliane; Striker, Gary; Riser, Bruce L; LaPage, Janine; Nast, Cynthia C

2002-07-01

mRNAs of pathogenetic importance in the development of diabetic nephropathy were measured in subjects with type 1 diabetes to determine whether these might be used to predict progression from normoalbuminuria to microalbuminuria. We proposed that conversion from normoalbuminuria to microalbuminuria would be most likely in subjects whose connective tissue growth factor (CTGF) and collagen mRNAs were above the 95% confidence interval (CI) for live renal donors and within the 95% CI for subjects with abnormal albuminuria. Glomerular CTGF, collagen alpha2(IV), and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured in microdissected glomeruli from living renal donors (n = 10), and subjects with normoalbuminuria (n = 12), microalbuminuria (n = 5), and overt proteinuria (n = 6). After 44 +/- 2 months of follow-up, one subject converted from normoalbuminuria to microalbuminuria. Although the data are limited, progression from normoalbuminuria to microalbuminuria occurred in the only normoalbuminuric subject whose mRNA levels were above the live renal donors' 95% CI for CTGF and collagen alpha2(IV) and within the 95% CI of subjects with abnormal albuminuria. No clinical or histopathologic finding distinguished the progressor from the nonprogressors at the time of biopsy. This case report provides proof-of-principle that a panel of glomerular mRNA markers chosen because of their pathogenetic relevance may be useful adjuncts to albuminuria and histology in predicting clinical stability or clinical progression in diabetic nephropathy. Copyright 2002 by the National Kidney Foundation, Inc.

Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

PubMed Central

Rubin, H; Salem, J S; Li, L S; Yang, F D; Mama, S; Wang, Z M; Fisher, A; Hamann, C S; Cooperman, B S

1993-01-01

Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle. Images Fig. 2 Fig. 4 Fig. 5 PMID:8415692

Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

PubMed Central

Winteler, H V; Schneidinger, B; Jaeger, K E; Haas, D

1996-01-01

The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase. PMID:8795231

Dynamic changes in gene expression during human trophoblast differentiation.

PubMed

Handwerger, Stuart; Aronow, Bruce

2003-01-01

The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

PubMed Central

Moser, Joanna J.; Fritzler, Marvin J.

2010-01-01

Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies. PMID:20976148

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

PubMed

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

PubMed

Feng, Ling; Wang, Ru; Lian, Meng; Ma, Hongzhi; He, Ning; Liu, Honggang; Wang, Haizhou; Fang, Jugao

2016-01-01

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

PubMed

Amiche, M; Ducancel, F; Lajeunesse, E; Boulain, J C; Ménez, A; Nicolas, P

1993-03-31

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

MicroRNA biogenesis and function in plants.

PubMed

Chen, Xuemei

2005-10-31

A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression

PubMed Central

Gomez, Javier A; Rutkowski, D Thomas

2016-01-01

Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation—an outcome that could be relevant to conditions such as metabolic syndrome. DOI: http://dx.doi.org/10.7554/eLife.20390.001 PMID:27938665

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

PubMed

Ávila-Mendoza, José; Pérez-Rueda, Ernesto; Urban-Sosa, Valeria; Carranza, Martha; Martínez-Moreno, Carlos G; Luna, Maricela; Arámburo, Carlos

2018-01-01

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH 2 ), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

PubMed Central

Balagopal, Vidya; Beemon, Karen L.

2017-01-01

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

PubMed

Alkallas, Rached; Fish, Lisa; Goodarzi, Hani; Najafabadi, Hamed S

2017-10-13

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."

ADAR RNA editing below the backbone.

PubMed

Keegan, Liam; Khan, Anzer; Vukic, Dragana; O'Connell, Mary

2017-09-01

ADAR RNA editing enzymes ( a denosine d e a minases acting on R NA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster , which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain-RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems. © 2017 Keegan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Bombesin-like peptides and receptors in normal fetal baboon lung: roles in lung growth and maturation.

PubMed

Emanuel, R L; Torday, J S; Mu, Q; Asokananthan, N; Sikorski, K A; Sunday, M E

1999-11-01

Previously, we have shown that bombesin-like peptide (BLP) promotes fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60 (ED60), peaked on approximately ED90, and then declined before term (ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs detected from ED125 until birth. BLP (0.1-10 nM) stimulated type II cell differentiation in organ cultures as assessed by [(3)H]choline incorporation into surfactant phospholipids, electron microscopy, and increased surfactant protein (SP) A- and/or SP-C-immunopositive cells and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60. Cell proliferation was induced by GRP, peaking on ED90. Similarly, blocking BLP degradation stimulated lung growth and maturation, which was completely reversed by a BLP-specific antagonist. The dissociation between GRP and GRP-receptor gene expression during ontogeny suggests that novel BLP receptors and/or peptides might be implicated in these responses.

Transcriptional dynamics with time-dependent reaction rates

NASA Astrophysics Data System (ADS)

Nandi, Shubhendu; Ghosh, Anandamohan

2015-02-01

Transcription is the first step in the process of gene regulation that controls cell response to varying environmental conditions. Transcription is a stochastic process, involving synthesis and degradation of mRNAs, that can be modeled as a birth-death process. We consider a generic stochastic model, where the fluctuating environment is encoded in the time-dependent reaction rates. We obtain an exact analytical expression for the mRNA probability distribution and are able to analyze the response for arbitrary time-dependent protocols. Our analytical results and stochastic simulations confirm that the transcriptional machinery primarily act as a low-pass filter. We also show that depending on the system parameters, the mRNA levels in a cell population can show synchronous/asynchronous fluctuations and can deviate from Poisson statistics.

A-to-I editing of coding and non-coding RNAs by ADARs

PubMed Central

Nishikura, Kazuko

2016-01-01

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference. PMID:26648264

Estrogen Receptor Mutants/Variants in Human Breast Cancer.

DTIC Science & Technology

1995-12-01

Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker dystrophies . Cell 1990, 63:1239-1248 4 Patriotis C, Makris A...the most common inherited disorder of platelets, Aspartylblucosaminuria, 2 an inherited lysosomal storage disorder, Duchenne and Becker muscular... dystrophies 3 or cancer progression.4m 5 Several estrogen receptor (ER) variant mRNAs have also been identified in human breast cancer biopsies.6,7,8, 9

Genome wide identification of Staufen2-bound mRNAs in embryonic rat brains.

PubMed

Maher-Laporte, Marjolaine; DesGroseillers, Luc

2010-05-01

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Staufen2 is an mRNA-binding protein expressed in the cell bodies and cellular processes of different brain cells. It is notably involved in the transport of dendritic mRNAs along microtubules. Its knockdown expression was shown to change spine morphology and impair synaptic functions. However, the identity of Staufen2-bound mRNAs in brain cells is still completely unknown. As a mean to identify these mRNAs, we immunoprecipitated Staufen2-containing mRNPs from embryonic rat brains and used a genome wide approach to identify Staufen2-associated mRNAs. The genome wide approach identified 1780 mRNAs in Staufen2-containing mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. These results represent an additional and important step in the characterization of Staufen2- mediated neuronal functions in rat brains.

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

PubMed

Liang, Guanxiang; Malmuthuge, Nilusha; Bao, Hua; Stothard, Paul; Griebel, Philip J; Guan, Le Luo

2016-08-11

Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Neuronal 3',3,5-triiodothyronine (T3) uptake and behavioral phenotype of mice deficient in Mct8, the neuronal T3 transporter mutated in Allan-Herndon-Dudley syndrome.

PubMed

Wirth, Eva K; Roth, Stephan; Blechschmidt, Cristiane; Hölter, Sabine M; Becker, Lore; Racz, Ildiko; Zimmer, Andreas; Klopstock, Thomas; Gailus-Durner, Valerie; Fuchs, Helmut; Wurst, Wolfgang; Naumann, Thomas; Bräuer, Anja; de Angelis, Martin Hrabé; Köhrle, Josef; Grüters, Annette; Schweizer, Ulrich

2009-07-29

Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan-Herndon-Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3',3,5-triiodothyronine (T(3)) plasma levels. Mice deficient in Mct8 replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lacking Mct8. Therefore, we subjected Mct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent on Mct8 are in a hypothyroid state, whereas neurons expressing other T(3) transporters become hyperthyroid, if they are exposed directly to the high plasma T(3). The majority of T(3) uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T(3) transporter classes. mRNAs encoding six T(3) transporters, including L-type amino acid transporters (LATs), were coexpressed with Mct8 in isolated neurons. We then demonstrated Lat2 expression in cultured neurons and throughout murine brain development. In contrast, LAT2 is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype in MCT8-deficient patients, whereas Mct8-deficient mouse neurons are functionally complemented by other transporters, for possibly Lat2.

Cigarette Smoking Decreases Global MicroRNA Expression in Human Alveolar Macrophages

PubMed Central

Graff, Joel W.; Powers, Linda S.; Dickson, Anne M.; Kim, Jongkwang; Reisetter, Anna C.; Hassan, Ihab H.; Kremens, Karol; Gross, Thomas J.

2012-01-01

Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory) and M2 (anti-inflammatory) polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an “inverse” M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages. PMID:22952876

Integrative analysis of long non-coding RNAs and messenger RNA expression profiles in systemic lupus erythematosus.

PubMed

Luo, Qing; Li, Xue; Xu, Chuxin; Zeng, Lulu; Ye, Jianqing; Guo, Yang; Huang, Zikun; Li, Junming

2018-03-01

Thousands of long noncoding RNAs (lncRNAs) have been reported and represent an important subset of pervasive genes associated with a broad range of biological functions. Abnormal expression levels of lncRNAs have been demonstrated in multiple types of human disease. However, the role of lncRNAs in systemic lupus erythematosus (SLE) remains poorly understood. In the present study, the expression patterns of lncRNAs and messenger RNAs (mRNAs) were investigated in peripheral blood mononuclear cells (PBMCs) in SLE using Human lncRNA Array v3.0 (8x60 K; Arraystar, Inc., Rockville, MD, USA). The microarray results indicated that 8,868 lncRNAs (3,657 upregulated and 5,211 downregulated) and 6,876 mRNAs (2,862 upregulated and 4,014 downregulated) were highly differentially expressed in SLE samples compared with the healthy group. Gene ontology (GO) analysis of lncRNA target prediction indicated the presence of 474 matched lncRNA‑mRNA pairs for 293 differentially expressed lncRNAs (fold change, ≥3.0) and 381 differentially expressed mRNAs (fold change, ≥3.0). The most enriched pathways were 'Transcriptional misregulation in cancer' and 'Valine, leucine and isoleucine degradation'. Furthermore, reverse transcription‑quantitative polymerase chain reaction data verified six abnormal lncRNAs and mRNAs in SLE. The results indicate that the lncRNA expression profile in SLE was significantly changed. In addition, a range of SLE‑associated lncRNAs were identified. Thus, the present results provide important insights regarding lncRNAs in the pathogenesis of SLE.

Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

PubMed

Simon, Jean; Milenkovic, Dragan; Godet, Estelle; Cabau, Cedric; Collin, Anne; Métayer-Coustard, Sonia; Rideau, Nicole; Tesseraud, Sophie; Derouet, Michel; Crochet, Sabine; Cailleau-Audouin, Estelle; Hennequet-Antier, Christelle; Gespach, Christian; Porter, Tom E; Duclos, Michel J; Dupont, Joëlle; Cogburn, Larry A

2012-03-01

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

PubMed Central

Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

2012-01-01

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

PubMed

Feliciani, Claudio; Toto, Paola; Wang, Binghe; Sauder, Daniel N; Amerio, Pierluigi; Tulli, Antonio

2003-08-01

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

Cytoskeletal actin genes function downstream of HNF-3beta in ascidian notochord development.

PubMed

Jeffery, W R; Ewing, N; Machula, J; Olsen, C L; Swalla, B J

1998-11-01

We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.

Tandemly arranged chalcone synthase A genes contribute to the spatially regulated expression of siRNA and the natural bicolor floral phenotype in Petunia hybrida.

PubMed

Morita, Yasumasa; Saito, Ryoko; Ban, Yusuke; Tanikawa, Natsu; Kuchitsu, Kazuyuki; Ando, Toshio; Yoshikawa, Manabu; Habu, Yoshiki; Ozeki, Yoshihiro; Nakayama, Masayoshi

2012-06-01

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections

PubMed Central

Sadek, Jouliana

2016-01-01

ABSTRACT During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5′ or 3′ end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections. IMPORTANCE Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections. PMID:27681125

C3aR and C5aR1 act as key regulators of human and mouse β-cell function.

PubMed

Atanes, Patricio; Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Hawkes, Ross; Liu, Bo; Zhao, Min; Huang, Guo Cai; Persaud, Shanta J; Amisten, Stefan

2018-02-01

Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca 2+ ]i), ATP generation and apoptosis were assessed by standard techniques. C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to β- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca 2+ ]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. Our observations demonstrate a functional link between activation of components of the innate immune system and improved β-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on β-cells.

Genes affecting novel seed constituents in Limnanthes alba Benth: transcriptome analysis of developing embryos and a new genetic map of meadowfoam

PubMed Central

Cooper, Laurel D.; Kishore, Venkata K.; Knapp, Steven J.; Kling, Jennifer G.

2015-01-01

The seed oil of meadowfoam, a new crop in the Limnanthaceae family, is highly enriched in very long chain fatty acids that are desaturated at the Δ5 position. The unusual oil is desirable for cosmetics and innovative industrial applications and the seed meal remaining after oil extraction contains glucolimnanthin, a methoxylated benzylglucosinolate whose degradation products are herbicidal and anti-microbial. Here we describe EST analysis of the developing seed transcriptome that identified major genes involved in biosynthesis and assembly of the seed oil and in glucosinolate metabolic pathways. mRNAs encoding acyl-CoA Δ5 desaturase were notably abundant. The library was searched for simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). Fifty-four new SSR markers and eight candidate gene markers were developed and combined with previously developed SSRs to construct a new genetic map for Limnanthes alba. Mapped genes in the lipid biosynthetic pathway encode 3-ketoacyl-CoA synthase (KCS), Δ5 desaturase (Δ5DS), lysophosphatidylacyl-acyl transferase (LPAT), and acyl-CoA diacylglycerol acyl transferase (DGAT). Mapped genes in glucosinolate biosynthetic and degradation pathways encode CYP79A, myrosinase (TGG), and epithiospecifier modifier protein (ESM). The resources developed in this study will further the domestication and improvement of meadowfoam as an oilseed crop. PMID:26038713

Molecular cloning and expression of rat brain endopeptidase 3.4.24.16.

PubMed

Dauch, P; Vincent, J P; Checler, F

1995-11-10

We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.

Alternative Splicing of Barley Clock Genes in Response to Low Temperature

PubMed Central

Calixto, Cristiane P. G.; Simpson, Craig G.; Waugh, Robbie; Brown, John W. S.

2016-01-01

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement. PMID:27959947

Alternative Pre-mRNA Splicing in Mammals and Teleost Fish: A Effective Strategy for the Regulation of Immune Responses Against Pathogen Infection.

PubMed

Chang, Ming Xian; Zhang, Jie

2017-07-15

Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing provides an important source of transcriptome and proteome complexity through selectively joining different coding elements to form mRNAs, which encode proteins with similar or distinct functions. In mammals, previous studies have shown the role of alternative splicing in regulating the function of the immune system, especially in the regulation of T-cell activation and function. As lower vertebrates, teleost fish mainly rely on a large family of pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) from various invading pathogens. In this review, we summarize recent advances in our understanding of alternative splicing of piscine PRRs including peptidoglycan recognition proteins (PGRPs), nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and their downstream signaling molecules, compared to splicing in mammals. We also discuss what is known and unknown about the function of splicing isoforms in the innate immune responses against pathogens infection in mammals and teleost fish. Finally, we highlight the consequences of alternative splicing in the innate immune system and give our view of important directions for future studies.

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

PubMed Central

Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T.; Tsao, Wei-chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A.; Aubé, Jeffrey; Xu, Liang

2015-01-01

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. PMID:25750985

Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor

PubMed Central

Morita, Miyo Terao; Tanaka, Yoshiyuki; Kodama, Takashi S.; Kyogoku, Yoshimasa; Yanagi, Hideki; Yura, Takashi

1999-01-01

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock σ-factor σ32 encoded by the rpoH gene. Increased σ32 levels result from both enhanced synthesis and stabilization. Previous work indicated that σ32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5′-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of σ32 synthesis further, we analyzed expression of rpoH–lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA–30S ribosome–tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator. PMID:10090722

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

PubMed Central

Liu, Donglin; Brockman, J. Michael; Dass, Brinda; Hutchins, Lucie N.; Singh, Priyam; McCarrey, John R.; MacDonald, Clinton C.; Graber, Joel H.

2007-01-01

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis. PMID:17158511

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

PubMed Central

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V.

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm. PMID:21948791

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage.

PubMed

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.

Implant materials generate different peri-implant inflammatory factors: poly-ether-ether-ketone promotes fibrosis and microtextured titanium promotes osteogenic factors.

PubMed

Olivares-Navarrete, Rene; Hyzy, Sharon L; Slosar, Paul J; Schneider, Jennifer M; Schwartz, Zvi; Boyan, Barbara D

2015-03-15

An in vitro study examining factors produced by human mesenchymal stem cells on spine implant materials. The aim of this study was to examine whether the inflammatory microenvironment generated by cells on titanium-aluminum-vanadium (Ti-alloy, TiAlV) surfaces is affected by surface microtexture and whether it differs from that generated on poly-ether-ether-ketone (PEEK). Histologically, implants fabricated from PEEK have a fibrous connective tissue surface interface whereas Ti-alloy implants demonstrate close approximation with surrounding bone. Ti-alloy surfaces with complex micron/submicron scale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation whereas PEEK favors fibrous tissue formation. Human mesenchymal stem cells were cultured on tissue culture polystyrene, PEEK, smooth TiAlV, or macro-/micro-/nano-textured rough TiAlV (mmnTiAlV) disks. Osteoblastic differentiation and secreted inflammatory interleukins were assessed after 7 days. Fold changes in mRNAs for inflammation, necrosis, DNA damage, or apoptosis with respect to tissue culture polystyrene were measured by low-density polymerase chain reaction array. Data were analyzed by analysis of variance, followed by Bonferroni's correction of Student's t-test. Cells on PEEK upregulated mRNAs for chemokine ligand-2, interleukin (IL) 1β, IL6, IL8, and tumor necrosis factor. Cells grown on the mmnTiAlV had an 8-fold reduction in mRNAs for toll-like receptor-4. Cells grown on mmnTiAlV had reduced levels of proinflammatory interleukins. Cells on PEEK had higher mRNAs for factors strongly associated with cell death/apoptosis, whereas cells on mmnTiAlV exhibited reduced cytokine factor levels. All results were significant (P < 0.05). These results suggest that fibrous tissue around PEEK implants may be due to several factors: reduced osteoblastic differentiation of progenitor cells and production of an inflammatory environment that favors cell death via apoptosis and necrosis. Ti alloy surfaces with complex macro/micro/nanoscale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation. N/A.

Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery.

PubMed

Donovan, Jesse; Rath, Sneha; Kolet-Mandrikov, David; Korennykh, Alexei

2017-11-01

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest. © 2017 Donovan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

PubMed Central

Bélanger, François; Stepinski, Janusz; Darzynkiewicz, Edward; Pelletier, Jerry

2010-01-01

Cellular eukaryotic mRNAs are capped at their 5′ ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2′-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2′-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo. PMID:20713356

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

PubMed Central

Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-01-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

PubMed

Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-12-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Human Interferon Regulatory Factor 2 Gene Expression is Induced in Chronic Hepatitis C Virus Infection—A Possible Mode of Viral Persistence

PubMed Central

Mukherjee, Rathindra M; Bansode, Budhapriyavilas; Gangwal, Puja; Jakkampudi, Aparna; Reddy, Panyala B; Rao, Padaki N; Gupta, Rajesh; Reddy, D Nageshwar

2012-01-01

Background The interferon regulatory factors (IRFs) are a family of transcription factors known to be involved in the modulation of cellular responses to interferons (IFNs) and viral infection. While IRF-1 acts as a positive regulator, IRF-2 is known to repress IFN-mediated gene expression. The increase in the IRF-1/IRF-2 ratio is considered as an important event in the transcriptional activation of IFN-α gene toward development of the cellular antiviral response. Objective This study was performed to assess the expression of IRF mRNAs along with the expression level of IFN-α, its receptor (IFNAR-1), and the signal transduction factor (STAT-1) in treatment naive hepatitis C virus (HCV)-infected subjects. Materials Thirty-five chronically infected (CHC) patients and 39 voluntary blood donors as controls were included in the study. Quantification of HCV-RNA (ribonucleic acid) and genotyping were done by real-time polymerase chain reaction (PCR) and hybridization assays, respectively, using patient's serum/plasma. In both controls and patients, the serum level of IFN-α and IFN-α was measured by flow cytometry. Target gene expressions were studied by retro-transcription of respective mRNAs extracted from peripheral blood mononuclear cells (PBMCs) followed by PCR amplification and densitometry. Minus-strand HCV-RNA as a marker of viral replication in PBMCs was detected by an inhouse PCR assay. Results Both IRF-1 and IRF-2 genes were significantly enhanced in CHC than in control subjects (P < 0.001). A significant positive correlation (r2 = 0.386, P <0.01) was obtained between higher IRF-2 gene expression and increasing level of HCV-RNA. Chronically infected subjects (13%) harboring replicating HCV in PBMCs showed no significant differences in gene expressions than the subjects without HCV in PBMCs. Conclusion Our findings indicate that HCV modulates host immunity by inducing IRF-2 gene to counteract IRF-1-mediated IFN-α gene expression. Since the IRF-2 gene is known to encode oncogenic protein, the role of IRF-2 in CHC patients developing hepatocellular carcinoma warrants further studies. PMID:25755403

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

PubMed

Lee, Y L; Lee, K F; Xu, J S; Kwok, K L; Luk, J M; Lee, W M; Yeung, W S B

2003-02-01

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

PubMed

Wang, H; Miao, S; Chen, D; Wang, L; Koide, S S

1999-10-06

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart.

PubMed

Geng, Zhimin; Wang, Jue; Pan, Lulu; Li, Ming; Zhang, Jitai; Cai, Xueli; Chu, Maoping

2017-04-01

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

PubMed

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A; Voorhoeve, P Mathijs

2012-05-01

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

PubMed Central

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A.; Voorhoeve, P. Mathijs

2012-01-01

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. PMID:22417692

Normal human gingival fibroblasts undergo cytostasis and apoptosis after long-term exposure to butyric acid.

PubMed

Shirasugi, Michihiro; Nishioka, Keisuke; Yamamoto, Toshiro; Nakaya, Takaaki; Kanamura, Narisato

2017-01-22

The causes of periodontal disease are complex. Butyric acid, a metabolite of periodontopathic bacteria such as Porphyromonas gingivalis, acts as a histone deacetylase inhibitor that has a direct effect on mRNA expression. Butyric acid produced by Clostridium butyricum in the intestinal tract induces differentiation of regulatory T cells, thereby suppressing inflammation in the gut. Mice lacking Clostridium butyricum in the intestinal tract suffer from colitis. By contrast, butyric acid in the oral cavity worsens periodontal disease. Periodontal disease is a chronic condition in which periodontal tissue is exposed to virulence factors (such as butyric acid); however, no study has examined the effects of long-term exposure to butyric acid. The present study demonstrated that long-term exposure of human gingival fibroblasts (HGFs) to butyric acid induced cytostasis and apoptosis via the intrinsic and extrinsic pathways. Butyric acid inhibited the division of HGFs by altering expression of mRNAs encoding cyclins. Butyric acid induced apoptosis in HGFs via the intrinsic pathway, followed by activation of caspase 9; there was no DNA damage or p53 activation. Butyric acid also upregulated expression of TNF-α mRNA and protein by HGFs. Furthermore TNF-α induced apoptosis by activating caspase 8 (the extrinsic pathway) and by inducing production of pro-inflammatory cytokines. Taken together, the results show that butyric acid induced cytostasis and apoptosis in HGFs, accompanied by production of pro-inflammatory cytokines. It thus acts as a death ligand and plays a critical role as a prophlogistic substance. Copyright © 2016 Elsevier Inc. All rights reserved.

A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

PubMed

Wong, J M; Ingles, C J

2001-02-01

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

Identification of genes differentially expressed in association with acquired cisplatin resistance

PubMed Central

Johnsson, A; Zeelenberg, I; Min, Y; Hilinski, J; Berry, C; Howell, S B; Los, G

2000-01-01

The goal of this study was to identify genes whose mRNA levels are differentially expressed in human cells with acquired cisplatin (cDDP) resistance. Using the parental UMSCC10b head and neck carcinoma cell line and the 5.9-fold cDDP-resistant subline, UMSCC10b/Pt-S15, two suppressive subtraction hybridization (SSH) cDNA libraries were prepared. One library represented mRNAs whose levels were increased in the cDDP resistant variant (the UP library), the other one represented mRNAs whose levels were decreased in the resistant cells (the DOWN library). Arrays constructed with inserts recovered from these libraries were hybridized with SSH products to identify truly differentially expressed elements. A total of 51 cDNA fragments present in the UP library and 16 in the DOWN library met the criteria established for differential expression. The sequences of 87% of these cDNA fragments were identified in Genbank. Among the mRNAs in the UP library that were frequently isolated and that showed high levels of differential expression were cytochrome oxidase I, ribosomal protein 28S, elongation factor 1α, α-enolase, stathmin, and HSP70. The approach taken in this study permitted identification of many genes never before linked to the cDDP-resistant phenotype. © 2000 Cancer Research Campaign PMID:10993653

Drosophila Pumilio Protein Contains Multiple Autonomous Repression Domains That Regulate mRNAs Independently of Nanos and Brain Tumor

PubMed Central

Weidmann, Chase A.

2012-01-01

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains. PMID:22064486

Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.

PubMed

Weidmann, Chase A; Goldstrohm, Aaron C

2012-01-01

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

PubMed Central

Diot, Cédric; Fournier, Guillaume; Dos Santos, Mélanie; Magnus, Julie; Komarova, Anastasia; van der Werf, Sylvie; Munier, Sandie; Naffakh, Nadia

2016-01-01

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19. PMID:27653209

Expression Signatures of Long Noncoding RNAs in Adolescent Idiopathic Scoliosis

PubMed Central

Wang, Liang; Yu, Bin; Zhuang, Qian-yu; Wang, Yi-Peng

2015-01-01

Purpose. Adolescent idiopathic scoliosis (AIS), the most common pediatric spinal deformity, is considered a complex genetic disease. Causing genes and pathogenesis of AIS are still unclear. This study was designed to identify differentially expressed long noncoding RNAs (lncRNAs) involving the pathogenesis of AIS. Methods. We first performed comprehensive screening of lncRNA and mRNA in AIS patients and healthy children using Agilent human lncRNA + mRNA Array V3.0 microarray. LncRNAs expression in different AIS patients was further evaluated using quantitative PCR. Results. A total of 139 lncRNAs and 546 mRNAs were differentially expressed between AIS patients and healthy control. GO and Pathway analysis showed that these mRNAs might be involved in bone mineralization, neuromuscular junction, skeletal system morphogenesis, nucleotide and nucleic acid metabolism, and regulation of signal pathway. Four lncRNAs (ENST00000440778.1, ENST00000602322.1, ENST00000414894.1, and TCONS_00028768) were differentially expressed between different patients when grouped according to age, height, classification, severity of scoliosis, and Risser grade. Conclusions. This study demonstrates the abnormal expression of lncRNAs and mRNAs in AIS, and the expression of some lncRNAs was related to clinical features. This study is helpful for further understanding of lncRNAs in pathogenesis, treatment, and prognosis of AIS. PMID:26421281

Mechanical Tension Increases CCN2/CTGF Expression and Proliferation in Gingival Fibroblasts via a TGFβ-Dependent Mechanism

PubMed Central

Guo, Fen; Carter, David E.; Leask, Andrew

2011-01-01

Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours) using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFβ, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1) mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring); but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFβ by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFβ type I (ALK5) receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFβ, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin. PMID:21611193

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Cleavage and polyadenylation: Ending the message expands gene regulation

PubMed Central

Neve, Jonathan

2017-01-01

ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

The cytochrome oxidase subunit I and subunit III genes in Oenothera mitochondria are transcribed from identical promoter sequences

PubMed Central

Hiesel, Rudolf; Schobel, Werner; Schuster, Wolfgang; Brennicke, Axel

1987-01-01

Two loci encoding subunit III of the cytochrome oxidase (COX) in Oenothera mitochondria have been identified from a cDNA library of mitochondrial transcripts. A 657-bp sequence block upstream from the open reading frame is also present in the two copies of the COX subunit I gene and is presumably involved in homologous sequence rearrangement. The proximal points of sequence rearrangements are located 3 bp upstream from the COX I and 1139 bp upstream from the COX III initiation codons. The 5'-termini of both COX I and COX III mRNAs have been mapped in this common sequence confining the promoter region for the Oenothera mitochondrial COX I and COX III genes to the homologous sequence block. ImagesFig. 5. PMID:15981332

Protein kinase WNK3 regulates the neuronal splicing factor Fox-1.

PubMed

Lee, A-Young; Chen, Wei; Stippec, Steve; Self, Jon; Yang, Fan; Ding, Xiaojun; Chen, She; Juang, Yu-Chi; Cobb, Melanie H

2012-10-16

We report an action of the protein kinase WNK3 on the neuronal mRNA splicing factor Fox-1. Fox-1 splices mRNAs encoding proteins important in synaptic transmission and membrane excitation. WNK3, implicated in the control of neuronal excitability through actions on ion transport, binds Fox-1 and inhibits its splicing activity in a kinase activity-dependent manner. Phosphorylation of Fox-1 by WNK3 does not change its RNA binding capacity; instead, WNK3 increases the cytoplasmic localization of Fox-1, thereby suppressing Fox-1-dependent splicing. These findings demonstrate a role of WNK3 in RNA processing. Considering the implication of WNK3 and Fox-1 in disorders of neuronal development such as autism, WNK3 may offer a target for treatment of Fox-1-induced disease.

Enterovirus Control of Translation and RNA Granule Stress Responses.

PubMed

Lloyd, Richard E

2016-03-30

Enteroviruses such as poliovirus (PV) and coxsackievirus B3 (CVB3) have evolved several parallel strategies to regulate cellular gene expression and stress responses to ensure efficient expression of the viral genome. Enteroviruses utilize their encoded proteinases to take over the cellular translation apparatus and direct ribosomes to viral mRNAs. In addition, viral proteinases are used to control and repress the two main types of cytoplasmic RNA granules, stress granules (SGs) and processing bodies (P-bodies, PBs), which are stress-responsive dynamic structures involved in repression of gene expression. This review discusses these processes and the current understanding of the underlying mechanisms with respect to enterovirus infections. In addition, the review discusses accumulating data suggesting linkage exists between RNA granule formation and innate immune sensing and activation.

Mammalian iron metabolism and its control by iron regulatory proteins☆

PubMed Central

Anderson, Cole P.; Shen, Lacy; Eisenstein, Richard S.; Leibold, Elizabeth A.

2013-01-01

Cellular iron homeostasis is maintained by iron regulatory proteins 1 and 2 (IRP1 and IRP2). IRPs bind to iron-responsive elements (IREs) located in the untranslated regions of mRNAs encoding protein involved in iron uptake, storage, utilization and export. Over the past decade, significant progress has been made in understanding how IRPs are regulated by iron-dependent and iron-independent mechanisms and the pathological consequences of IRP2 deficiency in mice. The identification of novel IREs involved in diverse cellular pathways has revealed that the IRP–IRE network extends to processes other than iron homeostasis. A mechanistic understanding of IRP regulation will likely yield important insights into the basis of disorders of iron metabolism. This article is part of a Special Issue entitled: Cell Biology of Metals. PMID:22610083

Human beta-globin mRNAs that harbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II that have a cap-like structure at the 5' termini.

PubMed Central

Lim, S K; Maquat, L E

1992-01-01

Previous studies have demonstrated that nonsense codons within beta zero-thalassemic or in vitro-mutagenized human beta-globin transgenes result in the production of mRNAs that are degraded abnormally rapidly in the cytoplasm of murine erythroid cells. As a consequence, three RNA degradative intermediates are formed that lack sequences from either exon I or exons I and II. We show here that the intermediates, like the full-length mRNA from which they derive and the endogenous murine beta maj-globin mRNA, bind to the anticap monoclonal antibody H-20 in a way that is competed by the cap analogue m7G and eliminated by prior exposure to tobacco acid pyrophosphatase. Furthermore, the intermediates, like the two full-length mRNAs, are resistant to a 5'----3' exonuclease activity isolated from HeLa cell nuclei that degrades uncapped but not capped ribopolymers. Based on these observations, the intermediates appear to possess a structure that is indistinguishable from the cap at the 5' end of mRNA, i.e. a methylated nucleoside that is linked to the RNA by a 5'-5' phosphodiester bond. Detection of the intermediates during murine development was concomitant with detection of full-length thalassemic mRNA. Intermediate production appears to be influenced by RNA structure as indicated by the products that derive from a beta zero-thalassemic beta-globin transgene harboring a structural alteration (a 4 bp deletion) that was larger than any of those previously studied. Images PMID:1324170

[Effects of ethanol extract of Rhizome Pinelliae Preparata on intracellular pH value of human gastric adenocarcinoma cells].

PubMed

Zhang, Ci-an; Wu, Feng; Mao, Zhu-jun; Wei, Zhen; Li, Yong-jin; Wei, Pin-kang

2011-08-01

To observe the effects of ethanol extract of Rhizome Pinelliae Preparata on the intracellular pH value of human gastric cancer SGC7901 cells. After coculturing SGC7901 cells with ethanol extract of Rhizome Pinelliae Preparata (1, 0.5, 0.25 and 0.125 mg/mL), cell viability was evaluated by chromatometry with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. Intracellular pH value of SGC7901 cells was measured in the monolayer by using the pH-sensitive fluorescent probe 2,7-bis-(2-carboxyethyl)-5-carboxyfluorescein-acetoxymethyl ester. The extracellular pH value of culture medium was measured by a pH211 Calibration Check Microprocessor pH Meter. Half-inhibitory concentration (IC(50)) of ethanol extract culture to SGC7901 cells was decided by the MTT method and expressions of vacuolar-H(+)-ATPase (V-ATPase) and Na(+)/H(+) exchanger isoform 1 (NHE1) mRNAs were examined by the method of fluorescence quantitative-polymerase chain reaction after 72 h of drug treatment. Ethanol extract of Rhizome Pinelliae Preparata at different concentrations significantly inhibited the proliferation of SGC7901 cells, lowered the intracellular pH values and heightened the extracellular pH values. The IC(50) of 72 h culture was 0.5mg/mL and it inhibited the expressions of V-ATPase and NHE1 mRNAs. Ethanol extract of Rhizome Pinelliae Preparata can lower down the intracellular pH value of SGC7901 cells. The mechanism may be related to inhibiting the expressions of V-ATPase and NHE1 mRNAs.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-03-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress.

Drosophila germ granules are structured and contain homotypic mRNA clusters

PubMed Central

Trcek, Tatjana; Grosch, Markus; York, Andrew; Shroff, Hari; Lionnet, Timothée; Lehmann, Ruth

2015-01-01

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules. PMID:26242323

Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype

PubMed Central

Pierson, Christopher R.; Dulin-Smith, Ashley N.; Durban, Ashley N.; Marshall, Morgan L.; Marshall, Jordan T.; Snyder, Andrew D.; Naiyer, Nada; Gladman, Jordan T.; Chandler, Dawn S.; Lawlor, Michael W.; Buj-Bello, Anna; Dowling, James J.; Beggs, Alan H.

2012-01-01

X-linked myotubular myopathy (MTM) is a severe neuromuscular disease of infancy caused by mutations of MTM1, which encodes the phosphoinositide lipid phosphatase, myotubularin. The Mtm1 knockout (KO) mouse has a severe phenotype and its short lifespan (8 weeks) makes it a challenge to use as a model in the testing of certain preclinical therapeutics. Many MTM patients succumb early in life, but some have a more favorable prognosis. We used human genotype–phenotype correlation data to develop a myotubularin-deficient mouse model with a less severe phenotype than is seen in Mtm1 KO mice. We modeled the human c.205C>T point mutation in Mtm1 exon 4, which is predicted to introduce the p.R69C missense change in myotubularin. Hemizygous male Mtm1 p.R69C mice develop early muscle atrophy prior to the onset of weakness at 2 months. The median survival period is 66 weeks. Histopathology shows small myofibers with centrally placed nuclei. Myotubularin protein is undetectably low because the introduced c.205C>T base change induced exon 4 skipping in most mRNAs, leading to premature termination of myotubularin translation. Some full-length Mtm1 mRNA bearing the mutation is present, which provides enough myotubularin activity to account for the relatively mild phenotype, as Mtm1 KO and Mtm1 p.R69C mice have similar muscle phosphatidylinositol 3-phosphate levels. These data explain the basis for phenotypic variability among human patients with MTM1 p.R69C mutations and establish the Mtm1 p.R69C mouse as a valuable model for the disease, as its less severe phenotype will expand the scope of testable preclinical therapies. PMID:22068590

Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects.

PubMed

Zhubi, Adrian; Chen, Ying; Guidotti, Alessandro; Grayson, Dennis R

2017-11-01

Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged amounts of 5hmC present in these regions, suggests the possibility that DNMT1 interacts with and alters MECP2 binding properties to selected promoters. Comparisons between data obtained from the FC with CB studies showed some common themes between brain regions which are discussed. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium

PubMed Central

Whitaker, L.H.R.; Murray, A.A.; Matthews, R.; Shaw, G.; Williams, A.R.W.; Saunders, P.T.K.

2017-01-01

Abstract STUDY QUESTION What is the impact of administration of the selective progesterone receptor modulator (SPRM), ulipristal acetate (UPA) on the endometrium of women with fibroids? SUMMARY ANSWER UPA administration altered expression of sex-steroid receptors and progesterone-regulated genes and was associated with low levels of glandular and stromal cell proliferation. WHAT IS KNOWN ALREADY Administration of all SPRM class members results in PAEC (progesterone receptor modulator associated endometrial changes). Data on the impact of the SPRM UPA administration on endometrial sex-steroid receptor expression, progesterone (P)-regulated genes and cell proliferation are currently lacking. STUDY DESIGN SIZE, DURATION Observational study with histological and molecular analyses to delineate impact of treatment with UPA on endometrium. Endometrial samples (n = 9) were collected at hysterectomy from women aged 39 to 49 with uterine fibroids treated with UPA (oral 5 mg daily) for 9–12 weeks. Control proliferative (n = 9) and secretory (n = 9) endometrium from women aged 38–52 with fibroids were derived from institutional tissue archives. PARTICIPANTS/MATERIALS, SETTING, METHODS Study setting was a University Research Institute. Endometrial biopsies were collected with institutional ethical approval and written informed consent. Concentrations of mRNAs encoded by steroid receptors, P-regulated genes and factors in decidualised endometrium were quantified with qRT-PCR. Immunohistochemistry was employed for localization of progesterone (PR, PRB), androgen (AR), estrogen (ERα) receptors and expression of FOXO1, HAND2, HOXA10, PTEN homologue. Endometrial glandular and stromal cell proliferation was objectively quantified using Ki67. MAIN RESULTS AND THE ROLE OF CHANCE UPA induced morphological changes in endometrial tissue consistent with PAEC. A striking change in expression patterns of PR and AR was detected compared with either proliferative or secretory phase samples. There were significant changes in pattern of expression of mRNAs encoded by IGFBP-1, FOXO1, IL-15, HAND2, IHH and HOXA10 compared with secretory phase samples consistent with low agonist activity in endometrium. Expression of mRNA encoded by FOXM1, a transcription factor implicated in cell cycle progression, was low in UPA-treated samples. Cell proliferation (Ki67 positive nuclei) was lower in samples from women treated with UPA compared with those in the proliferative phase. LARGE SCALE DATA N/A. LIMITATIONS REASONS FOR CAUTION A small number of well-characterized patients were studied in-depth. The impacts on morphology, molecular and cellular changes with SPRM, UPA administration on symptom control remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS P plays a pivotal role in endometrial function. P-action is mediated through interaction with the PR. These data provide support for onward development of the SPRM class of compounds as effective long-term medical therapy for heavy menstrual bleeding. STUDY FUNDING/COMPETING INTEREST(S) H.O.D.C. received has clinical research support for laboratory consumables and staff from Bayer Pharma Ag and provides consultancy advice (no personal remuneration) for Bayer Pharma Ag, PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc.; A.R.W.W. has received consultancy payments from Bayer, Gedeon Richter, Preglem SA, HRA Pharma; L.H.R.W., A.A.M., R.M., G.S. and P.T.K.S. have no conflicts of interest. Study funded in part from each of: Medical Research Council (G1002033; G1100356/1; MR/N022556/1); National Health Institute for Health Research (12/206/520) and TENOVUS Scotland. PMID:28130434

Visualization of RNA–protein interactions in living cells: FMRP and IMP1 interact on mRNAs

PubMed Central

Rackham, Oliver; Brown, Chris M

2004-01-01

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA–protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells. PMID:15282548

Insulin-like growth factors I and II in starry flounder (Platichthys stellatus): molecular cloning and differential expression during embryonic development.

PubMed

Xu, Yongjiang; Zang, Kun; Liu, Xuezhou; Shi, Bao; Li, Cunyu; Shi, Xueying

2015-02-01

In order to elucidate the possible roles of insulin-like growth factors I and II (IGF-I and IGF-II) in the embryonic development of Platichthys stellatus, their cDNAs were isolated and their spatial expression pattern in adult organs and temporal expression pattern throughout embryonic development were examined by quantitative real-time PCR assay. The IGF-I cDNA sequence was 1,268 bp in length and contained an open reading frame (ORF) of 558 bp, which encoded 185 amino acid residues. With respect to IGF-II, the full-length cDNA was 899 bp in length and contained a 648-bp ORF, which encoded 215 amino acid residues. The amino acid sequences of IGF-I and IGF-II exhibited high identities with their fish counterparts. The highest IGF-I mRNA level was found in the liver for both sexes, whereas the IGF-II gene was most abundantly expressed in female liver and male liver, gill, and brain. The sex-specific and spatial expression patterns of IGF-I and IGF-II mRNAs are thought to be related to the sexually dimorphic growth and development of starry flounder. Both IGF-I and IGF-II mRNAs were detected in unfertilized eggs, which indicated that IGF-I and IGF-II were parentally transmitted. Nineteen embryonic development stages were tested. IGF-I mRNA level remained high from unfertilized eggs to low blastula followed by a significant decrease at early gastrula and then maintained a lower level. In contrast, IGF-II mRNA level was low from unfertilized eggs to high blastula and peaked at low blastula followed by a gradual decrease. Moreover, higher levels of IGF-I mRNA than that of IGF-II were found from unfertilized eggs to high blastula, vice versa from low blastula to newly hatched larva, and the different expression pattern verified the differential roles of IGF-I and IGF-II in starry flounder embryonic development. These results could help in understanding the endocrine mechanism involved in the early development and growth of starry flounder.

Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

PubMed

Creelman, R A; Mullet, J E

1991-10-01

Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).

PubMed

Li, Jiuxuan; Zhang, Haibin; Zhang, Xiuyue; Yang, Shiyong; Yan, Taiming; Song, Zhaobin

2015-04-01

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant's schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5% identity and Sp-HSC70 shared more than 81.1% identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant's schizothoracin.

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

PubMed Central

Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V.

2014-01-01

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

Nucleotide sequence of RNA2 of Lettuce big-vein virus and evidence for a possible transcription termination/initiation strategy similar to that of rhabdoviruses.

PubMed

Sasaya, Takahide; Kusaba, Shinnosuke; Ishikawa, Koichi; Koganezawa, Hiroki

2004-09-01

Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210-1403 on the viral RNA), ORF2 (nt 1493-2494), ORF3 (nt 2617-3489), ORF4 (nt 3843-4337) and ORF5 (nt 4530-5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3' end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3'-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5'-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

Plant defense genes are regulated by ethylene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecker, J.R.; Davis, R.W.

One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (i and ii) genes that encode L-phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase (4-coumarate:CoA ligase (AMP-forming), EC 6.2.1.12), enzymes of the phenylpropanoid pathway, (iii) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (iv) the genesmore » encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.« less

A survey of small RNAs in human sperm

PubMed Central

Krawetz, Stephen A.; Kruger, Adele; Lalancette, Claudia; Tagett, Rebecca; Anton, Ester; Draghici, Sorin; Diamond, Michael P.

2011-01-01

BACKGROUND There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24 000 sncRNAs within each normal human spermatozoon. METHODS RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18–30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈7%), Piwi-interacting piRNAs (≈17%), repeat-associated small RNAs (≈65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization. PMID:21989093

Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

PubMed

Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

1992-01-01

This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.