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Sample records for aberrant mrna splicing

  1. Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

    PubMed

    Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

    2016-05-01

    Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

  2. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    PubMed Central

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks. PMID:24101931

  3. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM.

    PubMed

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-11-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

  4. Estimation of the minimum mRNA splicing error rate in vertebrates.

    PubMed

    Skandalis, A

    2016-01-01

    The majority of protein coding genes in vertebrates contain several introns that are removed by the mRNA splicing machinery. Errors during splicing can generate aberrant transcripts and degrade the transmission of genetic information thus contributing to genomic instability and disease. However, estimating the error rate of constitutive splicing is complicated by the process of alternative splicing which can generate multiple alternative transcripts per locus and is particularly active in humans. In order to estimate the error frequency of constitutive mRNA splicing and avoid bias by alternative splicing we have characterized the frequency of splice variants at three loci, HPRT, POLB, and TRPV1 in multiple tissues of six vertebrate species. Our analysis revealed that the frequency of splice variants varied widely among loci, tissues, and species. However, the lowest observed frequency is quite constant among loci and approximately 0.1% aberrant transcripts per intron. Arguably this reflects the "irreducible" error rate of splicing, which consists primarily of the combination of replication errors by RNA polymerase II in splice consensus sequences and spliceosome errors in correctly pairing exons.

  5. Aberrant and alternative splicing in skeletal system disease.

    PubMed

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy.

  6. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  7. Altered mRNA Splicing in SMN-Depleted Motor Neuron-Like Cells

    PubMed Central

    Todd, A. Gary; Astroski, Jacob W.; Lin, Hai; Liu, Yunlong

    2016-01-01

    Spinal muscular atrophy (SMA) is an intractable neurodegenerative disease afflicting 1 in 6–10,000 live births. One of the key functions of the SMN protein is regulation of spliceosome assembly. Reduced levels of the SMN protein that are observed in SMA have been shown to result in aberrant mRNA splicing. SMN-dependent mis-spliced transcripts in motor neurons may cause stresses that are particularly harmful and may serve as potential targets for the treatment of motor neuron disease or as biomarkers in the SMA patient population. We performed deep RNA sequencing using motor neuron-like NSC-34 cells to screen for SMN-dependent mRNA processing changes that occur following acute depletion of SMN. We identified SMN-dependent splicing changes, including an intron retention event that results in the production of a truncated Rit1 transcript. This intron-retained transcript is stable and is mis-spliced in spinal cord from symptomatic SMA mice. Constitutively active Rit1 ameliorated the neurite outgrowth defect in SMN depleted NSC-34 cells, while expression of the truncated protein product of the mis-spliced Rit1 transcript inhibited neurite extension. These results reveal new insights into the biological consequence of SMN-dependent splicing in motor neuron-like cells. PMID:27736905

  8. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-β -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing

    NASA Astrophysics Data System (ADS)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

    In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human β -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and β -globin sequences, we show that the NS1 5' splice site was effectively utilized by the β -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the β -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from β -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

  9. A synonymous CHRNE mutation responsible for an aberrant splicing leading to congenital myasthenic syndrome.

    PubMed

    Richard, Pascale; Gaudon, Karen; Fournier, Emmanuel; Jackson, Christopher; Bauché, Stéphanie; Haddad, Hafedh; Koenig, Jeanine; Echenne, Bernard; Hantaï, Daniel; Eymard, Bruno

    2007-05-01

    Congenital myasthenic syndromes (CMSs) are rare hereditary disorders transmitted in a recessive or dominant pattern, and are caused by mutations in the genes encoding proteins of the neuromuscular junction. They are classified in three groups depending on the origin of the molecular defect. Postsynaptic defects are the most frequent and have been reported to be partly due to abnormalities of the acetylcholine receptor, and particularly to mutations in CHRNE, the gene encoding the acetylcholine receptor epsilon-subunit. In a Portuguese patient with a mild form of recessive CMS, CHRNE sequencing identified an unknown homozygous transition. This variation affects the third nucleotide of the glycine 285 condon, and leads to a synonymous variant. Analysis of transcripts demonstrated that this single change creates a new splice donor site located 4 nucleotides upstream of the normal site, leading to a deletion and generating a frameshift in exon 9 followed by a premature termination codon. This paper relates the identification of a synonymous mutation in CHRNE that creates a new splice donor site leading to an aberrant splicing of pre-mRNAs and so to their instability. This is the first synonymous mutation in CHRNE known to generate a cryptic splice site, and mRNA quantification strongly suggests that it is the disease-causing mutation.

  10. Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

    PubMed

    Vetrini, Francesco; Tammaro, Roberta; Bondanza, Sergio; Surace, Enrico M; Auricchio, Alberto; De Luca, Michele; Ballabio, Andrea; Marigo, Valeria

    2006-05-01

    An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

  11. Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition.

    PubMed

    Královicová, Jana; Vorechovsky, Igor

    2007-01-01

    Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.

  12. Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

    PubMed

    Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

    2015-12-15

    We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

  13. Aberrant alternative splicing of thyroid hormone receptor in a TSH-secreting pituitary tumor is a mechanism for hormone resistance.

    PubMed

    Ando, S; Sarlis, N J; Krishnan, J; Feng, X; Refetoff, S; Zhang, M Q; Oldfield, E H; Yen, P M

    2001-09-01

    Patients with TSH-secreting pituitary tumors (TSHomas) have high serum TSH levels despite elevated thyroid hormone levels. The mechanism for this defect in the negative regulation of TSH secretion is not known. We performed RT-PCR to detect mutations in TRbeta from a surgically resected TSHoma. Analyses of the RT-PCR products revealed a 135-bp deletion within the sixth exon that encodes the ligand-binding domain of TRbeta2. This deletion was caused by alternative splicing of TRbeta2 mRNA, as near-consensus splice sequences were found at the junction site and no deletion or mutations were detected in the tumoral genomic DNA. This TRbeta variant (TRbeta2spl) lacked thyroid hormone binding and had impaired T3-dependent negative regulation of both TSHbeta and glycoprotein hormone alpha-subunit genes in cotransfection studies. Furthermore, TRbeta2spl showed dominant negative activity against the wild-type TRbeta2. These findings strongly suggest that aberrant alternative splicing of TRbeta2 mRNA generated an abnormal TR protein that accounted for the defective negative regulation of TSH in the TSHoma. This is the first example of aberrant alternative splicing of a nuclear hormone receptor causing hormonal dysregulation. This novel posttranscriptional mechanism for generating abnormal receptors may occur in other hormone-resistant states or tumors in which no receptor mutation is detected in genomic DNA.

  14. Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice.

    PubMed

    Oana, Kosuke; Oma, Yoko; Suo, Satoshi; Takahashi, Masanori P; Nishino, Ichizo; Takeda, Shin'ichi; Ishiura, Shoichi

    2013-01-01

    Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.

  15. Aberrant alternative splicing pattern of ADAR2 downregulates adenosine-to-inosine editing in glioma.

    PubMed

    Li, Zhaohui; Tian, Yu; Tian, Nan; Zhao, Xingli; Du, Chao; Han, Liang; Zhang, Haishan

    2015-06-01

    Adenosine-to-inosine (A-to-I) RNA editing is the most common type of RNA editing in mammals, and is catalyzed by adenosine deaminases acting on RNA (ADARs). ADAR2 is the main enzyme responsible for A-to-I editing in humans, and A-to-I underediting at the glutamine (Q)/arginine (R) site of the glutamate receptor subunit B (GluR-B) is associated with the pathogenesis and invasiveness of glioma. The level of ADAR2 mRNA expression and the alternative splicing of the ADAR2 pre-mRNA both affect the catalytic activity of ADAR2. However, reports of ADAR2 mRNA expression in glioma are inconsistent. The mechanism regulating ADAR2 pre-mRNA splicing is also unknown. In this study, we explored the deregulation of A-to-I RNA editing in glioma. We confirmed the underediting at the Q/R site of GluR-B mRNA in the glioma cell lines U87, U251 and A172 compared with that in normal human astrocytes (NHAs) HA1800. However, we demonstrated with reverse transcription (RT-PCR) and quantitative PCR (qPCR) that the expression of ADAR2 mRNA was not significantly altered in the glioma cell lines. Three alternative splicing sites are utilized in the glioma cell lines and NHAs: the first, located between exons -1 and 1, causes the inclusion of exon 1a; the second causes the removal of exon 2, which encodes two double-stranded RNA-binding domains; and the third, located between exons 4 and 6, causes the inclusion of alternative exon 5a, introducing a 120-nucleotide coding Alu-repeat sequence in frame. However, the expression ratio of two types of transcripts (with and without exon 5a) was altered in the glioma cells. Transcripts with exon 5a, which generate an ADAR2 isoform with ~50% reduced activity, were predominantly expressed in the glioma cell lines, whereas transcripts without exon 5a were predominantly expressed in the NHAs. From these results, we conclude that this aberrant alternative splicing pattern of ADAR2 downregulates A-to-I editing in glioma.

  16. DBASS3 and DBASS5: databases of aberrant 3'- and 5'-splice sites.

    PubMed

    Buratti, Emanuele; Chivers, Martin; Hwang, Gyulin; Vorechovsky, Igor

    2011-01-01

    DBASS3 and DBASS5 provide comprehensive repositories of new exon boundaries that were induced by pathogenic mutations in human disease genes. Aberrant 5'- and 3'-splice sites were activated either by mutations in the consensus sequences of natural exon-intron junctions (cryptic sites) or elsewhere ('de novo' sites). DBASS3 and DBASS5 currently contain approximately 900 records of cryptic and de novo 3'- and 5'-splice sites that were produced by over a thousand different mutations in approximately 360 genes. DBASS3 and DBASS5 data can be searched by disease phenotype, gene, mutation, location of aberrant splice sites in introns and exons and their distance from authentic counterparts, by bibliographic references and by the splice-site strength estimated with several prediction algorithms. The user can also retrieve reference sequences of both aberrant and authentic splice sites with the underlying mutation. These data will facilitate identification of introns or exons frequently involved in aberrant splicing, mutation analysis of human disease genes and study of germline or somatic mutations that impair RNA processing. Finally, this resource will be useful for fine-tuning splice-site prediction algorithms, better definition of auxiliary splicing signals and design of new reporter assays. DBASS3 and DBASS5 are freely available at http://www.dbass.org.uk/.

  17. PVAAS: identify variants associated with aberrant splicing from RNA-seq

    PubMed Central

    Wang, Liguo; Nie, Jinfu J.; Kocher, Jean-Pierre A.

    2015-01-01

    Motivation: RNA-seq has been widely used to study the transcriptome. Comparing to microarray, sequencing-based RNA-seq is able to identify splicing variants and single nucleotide variants in one experiment simultaneously. This provides unique opportunity to detect variants that associated with aberrant splicing. Despite the popularity of RNA-seq, no bioinformatics tool has been developed to leverage this advantage to identify variants associated with aberrant splicing. Results: We have developed PVAAS, a tool to identify single nucleotide variants that associated with aberrant alternative splicing from RNA-seq data. PVAAS works in three steps: (i) identify aberrant splicings; (ii) use user-provided variants or perform variant calling; (iii) assess the significance of association between variants and aberrant splicing events. Availability and implementation: PVAAS is written in Python and C. Source code and a comprehensive user’s manual are freely available at: http://pvaas.sourceforge.net/. Contact: wang.liguo@mayo.edu or kocher.jeanpierre@mayo.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573917

  18. The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

    PubMed Central

    Österman, Lennart; Lindsten, Hans; Holmberg, Monica

    2016-01-01

    Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML. PMID:27783661

  19. Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

    PubMed Central

    Ghosh, Anirban; Kuppusamy, Hemalatha; Pilarski, Linda M.

    2009-01-01

    Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression. PMID:19451652

  20. Regulation of hyaluronidase activity by alternative mRNA splicing.

    PubMed

    Lokeshwar, Vinata B; Schroeder, Grethchen L; Carey, Robert I; Soloway, Mark S; Iida, Naoko

    2002-09-13

    Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301-330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183-435 present in HYAL1 wild type), HYAL1v3 (aa 1-207), HYAL1v4 (aa 260-435), and HYAL1v5 (aa 340-435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299-328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251-417 present in HYAL3 wild type), and HYAL3v3 (aa 251-417, but lacking aa 299-328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer.

  1. Links between mRNA splicing, mRNA quality control, and intellectual disability

    PubMed Central

    Fasken, Milo B.; Corbett, Anita H.

    2016-01-01

    In recent years, the impairment of RNA binding proteins that play key roles in the post-transcriptional regulation of gene expression has been linked to numerous neurological diseases. These RNA binding proteins perform critical mRNA processing steps in the nucleus, including splicing, polyadenylation, and export. In many cases, these RNA binding proteins are ubiquitously expressed raising key questions about why only brain function is impaired. Recently, mutations in the ZC3H14 gene, encoding an evolutionarily conserved, polyadenosine RNA binding protein, have been linked to a nonsyndromic form of autosomal recessive intellectual disability. Thus far, research on ZC3H14 and its Nab2 orthologs in budding yeast and Drosophila reveals that ZC3H14/Nab2 is important for mRNA processing and neuronal patterning. Two recent studies now provide evidence that ZC3H14/Nab2 may function in the quality control of mRNA splicing and export and could help to explain the molecular defects that cause neuronal dysfunction and lead to an inherited form of intellectual disability. These studies on ZC3H14/Nab2 reveal new clues to the puzzle of why loss of the ubiquitously expressed ZC3H14 protein specifically affects neurons. PMID:27868086

  2. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    PubMed

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program.

  3. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  4. Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

    PubMed Central

    Barbazuk, W. Brad

    2017-01-01

    RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

  5. Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia

    PubMed Central

    2017-01-01

    The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too. PMID:28217708

  6. Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia.

    PubMed

    Zhou, Jianbiao; Chng, Wee-Joo

    2017-01-01

    The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too.

  7. A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia.

    PubMed

    Kubota, Tomoya; Roca, Xavier; Kimura, Takashi; Kokunai, Yosuke; Nishino, Ichizo; Sakoda, Saburo; Krainer, Adrian R; Takahashi, Masanori P

    2011-07-01

    Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5' splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5' splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect.

  8. Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

    PubMed Central

    Du, Hongqing; Cline, Melissa S.; Osborne, Robert J.; Tuttle, Daniel L.; Clark, Tyson A.; Donohue, John Paul; Hall, Megan P.; Shiue, Lily; Swanson, Maurice S.; Thornton, Charles A.; Ares, Manuel

    2009-01-01

    Myotonic dystrophy (DM1) is associated with expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. Strong correlation in splicing changes for ~100 new Mbnl1-regulated exons indicates loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA level changes can be attributed to loss of Mbnl1, indicating CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix (ECM) proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function, disrupting splicing, and loss of another function that disrupts ECM mRNA regulation, possibly mediated by MBNL2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies. PMID:20098426

  9. The RNA-binding protein QKI suppresses cancer-associated aberrant splicing.

    PubMed

    Zong, Feng-Yang; Fu, Xing; Wei, Wen-Juan; Luo, Ya-Ge; Heiner, Monika; Cao, Li-Juan; Fang, Zhaoyuan; Fang, Rong; Lu, Daru; Ji, Hongbin; Hui, Jingyi

    2014-04-01

    Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.

  10. Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling.

    PubMed

    Snyder, Chelsea A; Goodson, Michael L; Schroeder, Amy C; Privalsky, Martin L

    2015-09-15

    Alternative mRNA splicing diversifies the products encoded by the NCoR and SMRT corepressor loci. There is a programmed alteration in NCoR mRNA splicing during adipocyte differentiation from an NCoRω isoform, which contains three nuclear receptor interaction domains, to an NCoRδ isoform that contains two nuclear receptor interaction domains. This alternative mRNA splicing of NCoR has profound effects on adiposity and on diabetes in mouse models. We report here that dexamethasone, a powerful regulator of metabolism and of adipocyte differentiation, confers this change in NCoR mRNA splicing in cultured adipocytes. We also demonstrate that changes in dietary components can consistently, if moderately, modulate the total transcript levels and the mRNA splicing of NCoR and SMRT in both cultured cells and intact mice. This ability of alternative corepressor mRNA splicing to respond to nutritional changes confirms its importance in regulating glucose and lipid metabolism, and its promise as a therapeutic candidate for metabolic disorders such as type 2 diabetes.

  11. Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

    PubMed Central

    Snyder, Chelsea A.; Goodson, Michael L.; Schroeder, Amy C.; Privalsky, Martin L.

    2015-01-01

    Alternative mRNA splicing diversifies the products encoded by the NCoR and SMRT corepressor loci. There is a programmed alteration in NCoR mRNA splicing during adipocyte differentiation from an NCoRδ isoform, which contains three nuclear receptor interaction domains, to an NCoRδ isoform that contains two nuclear receptor interaction domains. This alternative mRNA splicing of NCoR has profound effects on adiposity and on diabetes in mouse models. We report here that dexamethasone, a powerful regulator of metabolism and of adipocyte differentiation, confers this change in NCoR mRNA splicing in cultured adipocytes. We also demonstrate that changes in dietary components can consistently, if moderately, modulate the total transcript levels and the mRNA splicing of NCoR and SMRT in both cultured cells and intact mice. This ability of alternative corepressor mRNA splicing to respond to nutritional changes confirms its importance in regulating glucose and lipid metabolism, and its promise as a therapeutic candidate for metabolic disorders such as type 2 diabetes. PMID:26166430

  12. Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues.

    PubMed

    Rinaldi, F; Terracciano, C; Pisani, V; Massa, R; Loro, E; Vergani, L; Di Girolamo, S; Angelini, C; Gourdon, G; Novelli, G; Botta, A

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a complex multisystemic disorder caused by an expansion of a CTG repeat located at the 3' untranslated region (UTR) of DMPK on chromosome 19q13.3. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms of DM1 including insulin resistance, muscle wasting and myotonia. In this paper we analyzed the expression of the MYH14 mRNA and protein in the muscle of DM1 patients (n=12) with different expansion lengths and normal subjects (n=7). The MYH14 gene is located on chromosome 19q13.3 and encodes for one of the heavy chains of the so called class II "nonmuscle" myosins (NMHCII). MYH14 has two alternative spliced isoforms: the inserted isoform (NMHCII-C1) which includes 8 amino acids located in the globular head of the protein, not encoded by the non inserted isoform (NMHCII-C0). Results showed a splicing unbalance of the MYH14 gene in DM1 muscle, with a prevalent expression of the NMHCII-C0 isoform more marked in DM1 patients harboring large CTG expansions. Minigene assay indicated that levels of the MBNL1 protein positively regulates the inclusion of the MYH14 exon 6. Quantitative analysis of the MYH14 expression revealed a significant reduction in the DM1 muscle samples, both at mRNA and protein level. No differences were found between DM1 and controls in the skeletal muscle localization of MYH14, obtained through immunofluorescence analysis. In line with the thesis of an "RNA gain of function" hypothesis described for the CTG mutation, we conclude that the alterations of the MYH14 gene may contribute to the DM1 molecular pathogenesis.

  13. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    PubMed Central

    Trochet, Delphine; Prudhon, Bernard; Jollet, Arnaud; Lorain, Stéphanie; Bitoun, Marc

    2016-01-01

    Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development. PMID:27623444

  14. Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients

    PubMed Central

    Sadakata, Tetsushi; Washida, Miwa; Iwayama, Yoshimi; Shoji, Satoshi; Sato, Yumi; Ohkura, Takeshi; Katoh-Semba, Ritsuko; Nakajima, Mizuho; Sekine, Yukiko; Tanaka, Mika; Nakamura, Kazuhiko; Iwata, Yasuhide; Tsuchiya, Kenji J.; Mori, Norio; Detera-Wadleigh, Sevilla D.; Ichikawa, Hironobu; Itohara, Shigeyoshi; Yoshikawa, Takeo; Furuichi, Teiichi

    2007-01-01

    Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder, and its underlying molecular mechanisms remain unknown. Ca2+-dependent activator protein for secretion 2 (CADPS2; also known as CAPS2) mediates the exocytosis of dense-core vesicles, and the human CADPS2 is located within the autism susceptibility locus 1 on chromosome 7q. Here we show that Cadps2-knockout mice not only have impaired brain-derived neurotrophic factor release but also show autistic-like cellular and behavioral phenotypes. Moreover, we found an aberrant alternatively spliced CADPS2 mRNA that lacks exon 3 in some autistic patients. Exon 3 was shown to encode the dynactin 1–binding domain and affect axonal CADPS2 protein distribution. Our results suggest that a disturbance in CADPS2-mediated neurotrophin release contributes to autism susceptibility. PMID:17380209

  15. Aberrant Splicing and Transcription Termination Caused by P Element Insertion into the Intron of a Drosophila Gene

    PubMed Central

    Horowitz, H.; Berg, C. A.

    1995-01-01

    Insertional mutagenesis screens using the P[lacZ, rosy(+)] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5' untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce a mRNA composed of 5' sequences from the mutated gene (in this case, pipsqueak) and 3' sequences from within the P[lacZ, rosy(+)] element. These truncated RNAs could yield proteins with dominant mutant effects. PMID:7705633

  16. Aberrant splicing and transcription termination caused by P element insertion into the intron of a Drosophila gene

    SciTech Connect

    Horowitz, H.; Berg, C.A.

    1995-01-01

    Insertional mutagenesis screens using the P[lacZ, rosy{sup +}] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5{prime} untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce an mRNA composed of 5{prime} sequences from the mutated gene (in this case, pipsqueak) and 3{prime} sequences from within the P[lacZ, rosy{sup +}] element. These truncated RNAs could yield proteins with dominant mutant effects. 43 refs., 4 figs.

  17. Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons.

    PubMed

    Szafranski, Karol; Fritsch, Claudia; Schumann, Frank; Siebel, Lisa; Sinha, Rileen; Hampe, Jochen; Hiller, Michael; Englert, Christoph; Huse, Klaus; Platzer, Matthias

    2014-08-01

    Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.

  18. Systematic analyses of rpm-1 suppressors reveal roles for ESS-2 in mRNA splicing in Caenorhabditis elegans.

    PubMed

    Noma, Kentaro; Goncharov, Alexandr; Jin, Yishi

    2014-11-01

    The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.

  19. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer

    PubMed Central

    Inoue, Kazushi; Fry, Elizabeth A.

    2015-01-01

    Breast cancer (BC) is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor) and human epidermal growth factor receptor 2 (HER2), play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER, HER2, and CD44 genes from the viewpoint of BC development. ERα36, a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2) has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms (CD44s, CD44v) play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci will provide keys

  20. An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

    PubMed Central

    Lin, Z; Wang, G; Demello, D E; Floros, J

    1999-01-01

    We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances. PMID:10493923

  1. Quantitative imaging of single mRNA splice variants in living cells

    NASA Astrophysics Data System (ADS)

    Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

    2014-06-01

    Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

  2. Mammalian mRNA Splice-Isoform Selection Is Tightly Controlled

    PubMed Central

    Chisa, Jennifer L.; Burke, David T.

    2007-01-01

    Post-transcriptional RNA processing is an important regulatory control mechanism for determining the phenotype of eukaryotic cells. The processing of a transcribed RNA species into alternative splice isoforms yields products that can perform different functions. Each type of cell in a multi-cellular organism is presumed to actively control the relative quantities of alternative splice isoforms. In this study, the alternatively spliced isoforms of five mRNA transcription units were examined by quantitative reverse transcription–PCR amplification. We show that interindividual variation in splice-isoform selection is very highly constrained when measured in a large population of genetically diverse mice (i.e., full siblings; N = 150). Remarkably, splice-isoform ratios are among the most invariant phenotypes measured in this population and are confirmed in a second, genetically distinct population. In addition, the patterns of splice-isoform selection show tissue-specific and age-related changes. We propose that splice-isoform selection is exceptionally robust to genetic and environmental variability and may provide a control point for cellular homeostasis. As a consequence, splice-isoform ratios may be useful as a practical quantitative measure of the physiological status of cells and tissues. PMID:17179090

  3. Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

    PubMed

    Shi, Lisong; Luo, Chunyan; Ahmed, Mairaj K; Attaie, Ali B; Ye, Xiaoqian

    2016-04-01

    Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

  4. Exploring the Pathogenic and Therapeutic Implications of Aberrant Splicing in Breast Cancer

    DTIC Science & Technology

    2011-07-01

    Shtivelman et al., 1985) and TMPRSS2‐ERG  found  in many  cases of  prostate   cancer   (Tomlins  et  al.,  2005).  New  efforts  using  high...of TMPRSS2 and ETS Transcription Factor Genes in  Prostate   Cancer . Science, 310(5748), 644‐648.  ...Pathogenic and Therapeutic Implications of Aberrant Splicing in Breast Cancer Dr. William Foulkes Sir Mortimer B. Davis- Jewish General Hospital

  5. Alternative splicing in cancer: implications for biology and therapy.

    PubMed

    Chen, J; Weiss, W A

    2015-01-02

    Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

  6. Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing

    SciTech Connect

    Freytag, S.O.; Beaudet, A.L.; Bock, H.G.O.; O'Brien, W.E.

    1984-10-01

    The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

  7. From polyadenylation to splicing: Dual role for mRNA 3' end formation factors.

    PubMed

    Misra, Ashish; Green, Michael R

    2016-01-01

    Recent genome-wide protein-RNA interaction studies have significantly reshaped our understanding of the role of mRNA 3' end formation factors in RNA biology. Originally thought to function solely in mediating cleavage and polyadenylation of mRNAs during their maturation, 3' end formation factors have now been shown to play a role in alternative splicing, even at internal introns--an unanticipated role for factors thought only to act at the 3' end of the mRNA. Here, we discuss the recent advances in our understanding of the role of 3' end formation factors in promoting global changes in alternative splicing at internal exon-intron junctions and how they act as cofactors for well known splicing regulators. Additionally, we review the mechanism by which these factors affect the recruitment of early intron recognition components to the 5' and 3' splice site. Our understanding of the roles of 3' end formation factors is still evolving, and the final picture might be more complex than originally envisioned.

  8. Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

    SciTech Connect

    Zhou, Z.; Olsen, J.C.; Silverman, L.M.

    1994-09-01

    Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. The remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.

  9. From General Aberrant Alternative Splicing in Cancers and Its Therapeutic Application to the Discovery of an Oncogenic DMTF1 Isoform

    PubMed Central

    Tian, Na; Li, Jialiang; Shi, Jinming; Sui, Guangchao

    2017-01-01

    Alternative pre-mRNA splicing is a crucial process that allows the generation of diversified RNA and protein products from a multi-exon gene. In tumor cells, this mechanism can facilitate cancer development and progression through both creating oncogenic isoforms and reducing the expression of normal or controllable protein species. We recently demonstrated that an alternative cyclin D-binding myb-like transcription factor 1 (DMTF1) pre-mRNA splicing isoform, DMTF1β, is increasingly expressed in breast cancer and promotes mammary tumorigenesis in a transgenic mouse model. Aberrant pre-mRNA splicing is a typical event occurring for many cancer-related functional proteins. In this review, we introduce general aberrant pre-mRNA splicing in cancers and discuss its therapeutic application using our recent discovery of the oncogenic DMTF1 isoform as an example. We also summarize new insights in designing novel targeting strategies of cancer therapies based on the understanding of deregulated pre-mRNA splicing mechanisms. PMID:28257090

  10. Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein

    SciTech Connect

    Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. )

    1992-03-03

    The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experiments show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.

  11. Regulation of mRNA splicing by MeCP2 via epigenetic modifications in the brain.

    PubMed

    Cheng, Tian-Lin; Chen, Jingqi; Wan, Huida; Tang, Bin; Tian, Weidong; Liao, Lujian; Qiu, Zilong

    2017-02-17

    Mutations of X-linked gene Methyl CpG binding protein 2 (MECP2) are the major causes of Rett syndrome (RTT), a severe neurodevelopmental disorder. Duplications of MECP2-containing genomic segments lead to severe autistic symptoms in human. MECP2-coding protein methyl-CpG-binding protein 2 (MeCP2) is involved in transcription regulation, microRNA processing and mRNA splicing. However, molecular mechanisms underlying the involvement of MeCP2 in mRNA splicing in neurons remain largely elusive. In this work we found that the majority of MeCP2-associated proteins are involved in mRNA splicing using mass spectrometry analysis with multiple samples from Mecp2-null rat brain, mouse primary neuron and human cell lines. We further showed that Mecp2 knockdown in cultured cortical neurons led to widespread alternations of mRNA alternative splicing. Analysis of ChIP-seq datasets indicated that MeCP2-regulated exons display specific epigenetic signatures, with DNA modification 5-hydroxymethylcytosine (5hmC) and histone modification H3K4me3 are enriched in down-regulated exons, while the H3K36me3 signature is enriched in exons up-regulated in Mecp2-knockdown neurons comparing to un-affected neurons. Functional analysis reveals that genes containing MeCP2-regulated exons are mainly involved in synaptic functions and mRNA splicing. These results suggested that MeCP2 regulated mRNA splicing through interacting with 5hmC and epigenetic changes in histone markers, and provide functional insights of MeCP2-mediated mRNA splicing in the nervous system.

  12. Regulation of mRNA splicing by MeCP2 via epigenetic modifications in the brain

    PubMed Central

    Cheng, Tian-Lin; Chen, Jingqi; Wan, Huida; Tang, Bin; Tian, Weidong; Liao, Lujian; Qiu, Zilong

    2017-01-01

    Mutations of X-linked gene Methyl CpG binding protein 2 (MECP2) are the major causes of Rett syndrome (RTT), a severe neurodevelopmental disorder. Duplications of MECP2-containing genomic segments lead to severe autistic symptoms in human. MECP2-coding protein methyl-CpG-binding protein 2 (MeCP2) is involved in transcription regulation, microRNA processing and mRNA splicing. However, molecular mechanisms underlying the involvement of MeCP2 in mRNA splicing in neurons remain largely elusive. In this work we found that the majority of MeCP2-associated proteins are involved in mRNA splicing using mass spectrometry analysis with multiple samples from Mecp2-null rat brain, mouse primary neuron and human cell lines. We further showed that Mecp2 knockdown in cultured cortical neurons led to widespread alternations of mRNA alternative splicing. Analysis of ChIP-seq datasets indicated that MeCP2-regulated exons display specific epigenetic signatures, with DNA modification 5-hydroxymethylcytosine (5hmC) and histone modification H3K4me3 are enriched in down-regulated exons, while the H3K36me3 signature is enriched in exons up-regulated in Mecp2-knockdown neurons comparing to un-affected neurons. Functional analysis reveals that genes containing MeCP2-regulated exons are mainly involved in synaptic functions and mRNA splicing. These results suggested that MeCP2 regulated mRNA splicing through interacting with 5hmC and epigenetic changes in histone markers, and provide functional insights of MeCP2-mediated mRNA splicing in the nervous system. PMID:28211484

  13. Alternative splicing in single cells dissected from complex tissues: separate expression of prepro-tachykinin A mRNA splice variants in sensory neurones.

    PubMed

    Springer, Jochen; McGregor, Gerard P; Fink, Ludger; Fischer, Axel

    2003-05-01

    Tachykinins play an important role in peripheral inflammatory diseases and disorders of the CNS. Most members of the tachykinin family are generated by alternative post-transcriptional splicing of the prepro-tachykinin (PPT) A gene. Here, we examined the simultaneous expression of PPT-A splice variants in individual neurones of the nodose ganglion. In extracts of ganglia, the expression of the four PPT-A mRNA splice variants and their four encoded peptides was shown by RT-PCR and combined HPLC and radioimmunoassay respectively. In order to examine prepro-tachykinin A expression in individual cells, single neurones were isolated from the ganglia using laser-assisted microdissection and processed for RT-PCR. Some 31.9% of the neurones investigated expressed a specific PPT-A transcript. Each individual neurone was found to express only a single splice variant. This is the first study to analyse the differential expression of PPT-A splice variants at the single-cell level. In view of the large number of alternatively spliced genes in the human genome and the resulting profound physiological effects, including several diseases, the technique described here is useful for isolating cells without possible confounding effects of dissociated neuronal cultures. For PPT-A, the results indicate that alternative post-transcriptional splicing determines the tachykinergic phenotype and may therefore have important functional implications.

  14. Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia.

    PubMed Central

    Pandolfi, P P; Alcalay, M; Fagioli, M; Zangrilli, D; Mencarelli, A; Diverio, D; Biondi, A; Lo Coco, F; Rambaldi, A; Grignani, F

    1992-01-01

    The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B-F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs. Images PMID:1314166

  15. Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases.

    PubMed

    Withers, Johanna B; Ashvetiya, Tamara; Beemon, Karen L

    2012-01-01

    Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3' half of the coding region. We were interested in examining splicing of the 5' half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20-40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

  16. Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

    PubMed Central

    Purcell, D F; Martin, M A

    1993-01-01

    Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity. Images PMID:8411338

  17. Analysis of aberrant splicing and nonsense-mediated decay of the stop codon mutations c.109G>T and c.504_505delCT in 7 patients with HMG-CoA lyase deficiency.

    PubMed

    Puisac, Beatriz; Teresa-Rodrigo, María Esperanza; Arnedo, María; Gil-Rodríguez, María Concepción; Pérez-Cerdá, Celia; Ribes, Antonia; Pié, Angeles; Bueno, Gloria; Gómez-Puertas, Paulino; Pié, Juan

    2013-04-01

    Eukaryotic cells can be protected against mutations that generate stop codons by nonsense-mediated mRNA decay (NMD) and/or nonsense-associated altered splicing (NAS). However, the processes are only partially understood and do not always occur. In this work, we study these phenomena in the stop codon mutations c.109G>T (p.Glu37*) and c.504_505delCT; the second and third most frequent mutations in HMG-CoA lyase deficiency (MIM #246450). The deficiency affects the synthesis of ketone bodies and produces severe disorders during early childhood. We used a minigene approach, real-time quantitative PCR and the inhibition of NMD by puromycin treatment, to study the effect of stop codons on splicing (NAS) and NMD in seven patients. Surprisingly, none of the stop codons studied appears to be the direct cause of aberrant splicing. In the mutation c.109G>T, the splicing is due to the base change G>T at position 109, which is critical and cannot be explained by disruption of exonic splicing enhancer (ESE) elements, by the appearance of exonic splicing silencer (ESS) elements which were predicted by bioinformatic tools or by the stop codons. Moreover, the mutation c.504_505delCT produces two mRNA transcripts both with stop codons that generate simultaneous NMD phenomena. The effects of the mutations studied on splicing seemed to be similar in all the patients. Furthermore, we report a Spanish patient with 3-hydroxy-3-methylglutaric aciduria and a novel missense mutation: c.825C>G (p.Asn275Lys).

  18. Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

    PubMed Central

    Yamashita, M; Krystal, M; Palese, P

    1988-01-01

    In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types. Images PMID:3404579

  19. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome.

    PubMed

    Finley, Jahahreeh

    2015-09-01

    Although the use of antiretroviral therapy (ART) has proven highly effective in controlling and suppressing HIV-1 replication, the persistence of latent but replication-competent proviruses in a small subset of CD4(+) memory T cells presents significant challenges to viral eradication from infected individuals. Attempts to eliminate latent reservoirs are epitomized by the 'shock and kill' approach, a strategy involving the combinatorial usage of compounds that influence epigenetic modulation and initiation of proviral transcription. However, efficient regulation of viral pre-mRNA splicing through manipulation of host cell splicing machinery is also indispensible for HIV-1 replication. Interestingly, aberrant alternative splicing of the LMNA gene via the usage of a cryptic splice site has been shown to be the cause of most cases of Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic condition characterized by an accelerated aging phenotype due to the accumulation of a truncated form of lamin A known as progerin. Recent evidence has shown that inhibition of the splicing factors ASF/SF2 (or SRSF1) and SRp55 (or SRSF6) leads to a reduction or an increase in progerin at both the mRNA and protein levels, respectively, thus altering the LMNA pre-mRNA splicing ratio. It is also well-established that during the latter stages of HIV-1 infection, an increase in the production and nuclear export of unspliced viral mRNA is indispensible for efficient HIV-1 replication and that the presence of ASF/SF2 leads to excessive viral pre-mRNA splicing and a reduction of unspliced mRNA, while the presence of SRp55 inhibits viral pre-mRNA splicing and aids in the generation and translation of unspliced HIV-1 mRNAs. The splicing-factor associated protein and putative mitochondrial chaperone p32 has also been shown to inhibit ASF/SF2, increase unspliced HIV-1 viral mRNA, and enhance mitochondrial DNA replication and oxidative phosphorylation. It is our hypothesis that activation of

  20. Ire1 Mediated mRNA Splicing in a C-Terminus Deletion Mutant of Drosophila Xbp1

    PubMed Central

    Coelho, Dina S.; Gaspar, Catarina J.; Domingos, Pedro M.

    2014-01-01

    The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER) stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2 - HR2 - and the C-terminal translational pausing site) present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. Here, we generated a Drosophila Xbp1 deletion mutant (Excision101) lacking both HR2 and C-terminal region, but not the Ire1 splicing site. We show that Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. Our results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced. PMID:25136861

  1. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

  2. Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

    PubMed Central

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

  3. A Novel Role for Protein Kinase Kin2 in Regulating HAC1 mRNA Translocation, Splicing, and Translation

    PubMed Central

    Anshu, Ashish; Mannan, M. Amin-ul; Chakraborty, Abhijit; Chakrabarti, Saikat

    2014-01-01

    A signaling network called the unfolded protein response (UPR) resolves the protein-folding defects in the endoplasmic reticulum (ER) from yeasts to humans. In the yeast Saccharomyces cerevisiae, the UPR activation involves (i) aggregation of the ER-resident kinase/RNase Ire1 to form an Ire1 focus, (ii) targeting HAC1 pre-mRNA toward the Ire1 focus that cleaves out an inhibitory intron from the mRNA, and (iii) translation of Hac1 protein from the spliced mRNA. Targeting HAC1 mRNA to the Ire1 focus requires a cis-acting bipartite element (3′BE) located at the 3′ untranslated leader. Here, we report that the 3′BE plays an additional role in promoting translation from the spliced mRNA. We also report that a high dose of either of two paralogue kinases, Kin1 and Kin2, overcomes the defective UPR caused by a mutation in the 3′BE. These results define a novel role for Kin kinases in the UPR beyond their role in cell polarity and exocytosis. Consistently, targeting, splicing, and translation of HAC1 mRNA are substantially reduced in the kin1Δ kin2Δ strain. Furthermore, we show that Kin2 kinase domain itself is sufficient to activate the UPR, suggesting that Kin2 initiates a signaling cascade to ensure an optimum UPR. PMID:25348718

  4. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery.

    PubMed

    Garanto, Alejandro; Chung, Daniel C; Duijkers, Lonneke; Corral-Serrano, Julio C; Messchaert, Muriël; Xiao, Ru; Bennett, Jean; Vandenberghe, Luk H; Collin, Rob W J

    2016-06-15

    Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.

  5. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    SciTech Connect

    Li, Long; Pintel, David J.

    2012-04-25

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  6. A Novel, Non-canonical Splice Variant of the Ikaros Gene Is Aberrantly Expressed in B-cell Lymphoproliferative Disorders

    PubMed Central

    Mancarelli, Maria Michela; Verzella, Daniela; Fischietti, Mariafausta; Di Tommaso, Ambra; Maccarone, Rita; Plebani, Sara; Di Ianni, Mauro; Gulino, Alberto; Alesse, Edoardo

    2013-01-01

    The Ikaros gene encodes a Krüppel-like zinc-finger transcription factor involved in hematopoiesis regulation. Ikaros has been established as one of the most clinically relevant tumor suppressors in several hematological malignancies. In fact, expression of dominant negative Ikaros isoforms is associated with adult B-cell acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia and adult and juvenile chronic myeloid leukemia. Here, we report the isolation of a novel, non-canonical Ikaros splice variant, called Ikaros 11 (Ik11). Ik11 is structurally related to known dominant negative Ikaros isoforms, due to the lack of a functional DNA-binding domain. Interestingly, Ik11 is the first Ikaros splice variant missing the transcriptional activation domain. Indeed, we demonstrated that Ik11 works as a dominant negative protein, being able to dimerize with Ikaros DNA-binding isoforms and inhibit their functions, at least in part by retaining them in the cytoplasm. Notably, we demonstrated that Ik11 is the first dominant negative Ikaros isoform to be aberrantly expressed in B-cell lymphoproliferative disorders, such as chronic lymphocytic leukemia. Aberrant expression of Ik11 interferes with both proliferation and apoptotic pathways, providing a mechanism for Ik11 involvement in tumor pathogenesis. Thus, Ik11 could represent a novel marker for B-cell lymphoproliferative disorders. PMID:23874502

  7. TGF-beta(1) regulation of human AT(1) receptor mRNA splice variants harboring exon 2.

    PubMed

    Martin, Mickey M; Buckenberger, Jessica A; Knoell, Daren L; Strauch, Arthur R; Elton, Terry S

    2006-04-25

    At least four alternatively spliced mRNAs can be synthesized from the human AT(1)R (hAT(1)R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT(1)R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT(1)R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT(1)R mRNA transcripts dramatically reduces hAT(1)R protein levels (nine-fold) and significantly attenuates Ang II responsiveness ( approximately four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT(1)R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT(1)R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-beta(1) treatment (4ng/ml for 4h) of fibroblasts up-regulated hAT(1)R mRNA splice variants, which harbored exon 2, six-fold. Since AT(1)R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT(1)R protein and possibly limit the potential pathological effects of AT(1)R activation.

  8. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

    PubMed Central

    Caminsky, Natasha; Mucaki, Eliseos J.; Rogan, Peter K.

    2014-01-01

    The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368

  9. Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

    PubMed

    Fournier, Guillaume; Chiang, Chiayn; Munier, Sandie; Tomoiu, Andru; Demeret, Caroline; Vidalain, Pierre-Olivier; Jacob, Yves; Naffakh, Nadia

    2014-06-01

    Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

  10. A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina.

    PubMed

    Prasov, Lev; Brown, Nadean L; Glaser, Tom

    2010-08-24

    The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3'RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

  11. An abnormal mRNA produced by a novel PMP22 splice site mutation associated with HNPP.

    PubMed

    Bellone, E; Balestra, P; Ribizzi, G; Schenone, A; Zocchi, G; Di Maria, E; Ajmar, F; Mandich, P

    2006-04-01

    Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating neuropathy. Point mutations in the PMP22 gene are a rare cause of HNPP. A novel PMP22 splice site mutation (c.179+1 G-->C) is reported in an HNPP family. By reverse transcriptase-polymerase chain reaction experiments, this mutation was shown to cause the synthesis of an abnormal mRNA in which a premature stop codon probably produces a truncated non-functional protein.

  12. Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants

    PubMed Central

    Vallée, Maxime P.; Di Sera, Tonya L.; Nix, David A.; Paquette, Andrew M.; Parsons, Michael T.; Bell, Russel; Hoffman, Andrea; Hogervorst, Frans B. L.; Goldgar, David E.; Spurdle, Amanda B.

    2016-01-01

    ABSTRACT Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs—the “integrated evaluation”—that combines a sequence analysis‐based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis‐based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice‐site fitness program MaxEntScan to generate spliceogenicity‐based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity‐based probability of pathogenicity for BRCA gene single‐nucleotide substitutions. We also updated the BRCA gene Ex‐UV LOVD, available at http://hci‐exlovd.hci.utah.edu, with 77 re‐evaluable variants. PMID:26913838

  13. During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

    PubMed Central

    Bailleul, B

    1996-01-01

    Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping. PMID:8604331

  14. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    SciTech Connect

    Morcos, Paul A. . E-mail: pmorcos@gene-tools.com

    2007-06-29

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a {beta}-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

  15. RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo.

    PubMed

    Tanaka, Naoko; Meineke, Birthe; Shuman, Stewart

    2011-09-02

    RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

  16. Applying genetic programming to the prediction of alternative mRNA splice variants.

    PubMed

    Vukusic, Ivana; Grellscheid, Sushma Nagaraja; Wiehe, Thomas

    2007-04-01

    Genetic programming (GP) can be used to classify a given gene sequence as either constitutively or alternatively spliced. We describe the principles of GP and apply it to a well-defined data set of alternatively spliced genes. A feature matrix of sequence properties, such as nucleotide composition or exon length, was passed to the GP system "Discipulus." To test its performance we concentrated on cassette exons (SCE) and retained introns (SIR). We analyzed 27,519 constitutively spliced and 9641 cassette exons including their neighboring introns; in addition we analyzed 33,316 constitutively spliced introns compared to 2712 retained introns. We find that the classifier yields highly accurate predictions on the SIR data with a sensitivity of 92.1% and a specificity of 79.2%. Prediction accuracies on the SCE data are lower, 47.3% (sensitivity) and 70.9% (specificity), indicating that alternative splicing of introns can be better captured by sequence properties than that of exons.

  17. Two novel exonic point mutations in HEXA identified in a juvenile Tay-Sachs patient: role of alternative splicing and nonsense-mediated mRNA decay.

    PubMed

    Levit, A; Nutman, D; Osher, E; Kamhi, E; Navon, R

    2010-06-01

    We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=), located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G (p.Asn399Asp) in exon 11, has been previously characterized as a common polymorphism in African-Americans. Hex A activity measured in TSD Glial cells, transfected with HEXA cDNA constructs bearing these mutations, was unaltered from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR products revealed three aberrant transcripts in the patient, one where exon 8 was absent, one where exon 11 was absent and a third lacking both exons 10 and 11. All three novel transcripts contain frameshifts resulting in premature termination codons (PTCs). Transfection of mini-gene constructs carrying the c.814G>A and c.1305C>T mutations proved that the two mutations result in exon skipping. mRNAs that harbor a PTC are detected and degraded by the nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal proteins. However, although NMD is functional in the patient's fibroblasts, aberrant transcripts are still present. We suggest that the level of correctly spliced transcripts as well as the efficiency in which NMD degrade the PTC-containing transcripts, apparently plays an important role in the phenotype severity of the unique patient and thus should be considered as a potential target for drug therapy.

  18. Influenza Viruses and mRNA Splicing: Doing More with Less

    PubMed Central

    Dubois, Julia

    2014-01-01

    ABSTRACT During their nuclear replication stage, influenza viruses hijack the host splicing machinery to process some of their RNA segments, the M and NS segments. In this review, we provide an overview of the current knowledge gathered on this interplay between influenza viruses and the cellular spliceosome, with a particular focus on influenza A viruses (IAV). These viruses have developed accurate regulation mechanisms to reassign the host spliceosome to alter host cellular expression and enable an optimal expression of specific spliced viral products throughout infection. Moreover, IAV segments undergoing splicing display high levels of similarity with human consensus splice sites and their viral transcripts show noteworthy secondary structures. Sequence alignments and consensus analyses, along with recently published studies, suggest both conservation and evolution of viral splice site sequences and structure for improved adaptation to the host. Altogether, these results emphasize the ability of IAV to be well adapted to the host’s splicing machinery, and further investigations may contribute to a better understanding of splicing regulation with regard to viral replication, host range, and pathogenesis. PMID:24825008

  19. Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA

    SciTech Connect

    Gutman, A.; Kornblihtt, A.R.

    1987-10-01

    The authors describe here a third region of variability in human fibronectin (FN) due to alternative RNA splicing. Two other positions of alternative splicing have been reported previously (ED and IIICS). The third region involves a 273-nucleotide exon encoding exactly one 91-amino acid repeat of type III homology, located between the DNA- and the cell-binding domains of FN, which is either included in or excluded from FN mRNA. The two mRNA variants arising by an exon-skipping mechanism are present in cells known to synthesize the cellular form of FN. However, liver cells, which are the source of plasma FN, produce only messengers without the extra type III sequence. Therefore, the region described here resembles, both structurally and functionally, the previously described ED (for extra domain) region, located toward the C terminus of the molecule between the cell- and heparin- (hep 2) binding domains. The authors conclude that both the extra type III repeat (names EDII) and ED represent sequences restricted to cellular FN. Combination of all the possible patterns of splicing in the three regions described to date may generate up to 20 distinct FN polypeptides from a single gene.

  20. Starvation actively inhibits splicing of glucose-6-phosphate dehydrogenase mRNA via a bifunctional ESE/ESS element bound by hnRNP K.

    PubMed

    Cyphert, T J; Suchanek, A L; Griffith, B N; Salati, L M

    2013-09-01

    Regulated expression of glucose-6-phosphate dehydrogenase (G6PD) is due to changes in the rate of pre-mRNA splicing and not changes in its transcription. Starvation alters pre-mRNA splicing by decreasing the rate of intron removal, leading to intron retention and a decrease in the accumulation of mature mRNA. A regulatory element within exon 12 of G6PD pre-mRNA controls splicing efficiency. Starvation caused an increase in the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and this increase coincided with the increase in the binding of hnRNP K to the regulatory element and a decrease in the expression of G6PD mRNA. HnRNP K bound to two C-rich motifs forming an ESS within exon 12. Overexpression of hnRNP K decreased the splicing and expression of G6PD mRNA, while siRNA-mediated depletion of hnRNP K caused an increase in the splicing and expression of G6PD mRNA. Binding of hnRNP K to the regulatory element was enhanced in vivo by starvation coinciding with a decrease in G6PD mRNA. HnRNP K binding to the C-rich motifs blocked binding of serine-arginine rich, splicing factor 3 (SRSF3), a splicing enhancer. Thus hnRNP K is a nutrient regulated splicing factor responsible for the inhibition of the splicing of G6PD during starvation.

  1. An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

    PubMed Central

    Widera, Marek; Erkelenz, Steffen; Hillebrand, Frank; Krikoni, Aikaterini; Widera, Darius; Kaisers, Wolfgang; Deenen, René; Gombert, Michael; Dellen, Rafael; Pfeiffer, Tanya; Kaltschmidt, Barbara; Münk, Carsten; Bosch, Valerie; Köhrer, Karl

    2013-01-01

    Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3′ splice site (3′ss) A1 but lack splicing at 5′ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3′ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3′ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5′ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5′ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. PMID:23255806

  2. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

    PubMed Central

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2014-01-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins. PMID:24213538

  3. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

    PubMed

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2013-12-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

  4. Early base-pair fluctuations and the activation of mRNA splicing

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    1991-05-01

    By means of multiprocessed Monte Carlo simulations we study the amplification in time structural fluctuations in sequential RNA folding concomitant with transcription. The simulations allow for an exploration of configuration space subject to the realistic time-constraints of RNA synthesis. The treatment focuses on the splicing YC4 intron as a study case. We show how an early disruption in the folding may result in a terminal structure which is active for splicing, bringing together the two cleavage sites at both ends of the intron.

  5. Impact of Alternative Initiation, Splicing, and Termination on the Diversity of the mRNA Transcripts Encoded by the Mouse Transcriptome

    PubMed Central

    Zavolan, Mihaela; Kondo, Shinji; Schönbach, Christian; Adachi, Jun; Hume, David A.; Hayashizaki, Yoshihide; Gaasterland, Terry

    2003-01-01

    We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleotide distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs. PMID:12819126

  6. Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Oma, Yoko; Hattori, Nobutaka; Ishiura, Shoichi; Nukina, Nobuyuki

    2015-02-01

    In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

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

    SciTech Connect

    Grzybowska, Ewa A.

    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-associated 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.

  8. Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia☆

    PubMed Central

    Hulse, R.P.; Beazley-Long, N.; Hua, J.; Kennedy, H.; Prager, J.; Bevan, H.; Qiu, Y.; Fernandes, E.S.; Gammons, M.V.; Ballmer-Hofer, K.; Gittenberger de Groot, A.C.; Churchill, A.J.; Harper, S.J.; Brain, S.D.; Bates, D.O.; Donaldson, L.F.

    2014-01-01

    Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event – leading to the preferential expression of VEGF-A165b over VEGF165a – prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. PMID:25151644

  9. Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

    PubMed

    Brummell, David A; Chen, Ronan K Y; Harris, John C; Zhang, Huaibi; Hamiaux, Cyril; Kralicek, Andrew V; McKenzie, Marian J

    2011-06-01

    Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.

  10. Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants

    PubMed Central

    Brummell, David A.; Chen, Ronan K. Y.; Harris, John C.; Zhang, Huaibi; Hamiaux, Cyril; Kralicek, Andrew V.; McKenzie, Marian J.

    2011-01-01

    Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability. PMID:21393382

  11. A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

    PubMed Central

    Drummond, Meghan C.; Friderici, Karen H.

    2013-01-01

    Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as “regulated unproductive splicing and translation” (RUST). PMID:24098136

  12. Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.

    PubMed

    Hull, J; Shackleton, S; Harris, A

    1994-07-01

    Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

  13. Faithful mRNA splicing depends on the Prp19 complex subunit faint sausage and is required for tracheal branching morphogenesis in Drosophila.

    PubMed

    Sauerwald, Julia; Soneson, Charlotte; Robinson, Mark D; Luschnig, Stefan

    2017-02-15

    Morphogenesis requires the dynamic regulation of gene expression, including transcription, mRNA maturation and translation. Dysfunction of the general mRNA splicing machinery can cause surprisingly specific cellular phenotypes, but the basis for these effects is not clear. Here, we show that the Drosophila faint sausage (fas) locus, which is implicated in epithelial morphogenesis and has previously been reported to encode a secreted immunoglobulin domain protein, in fact encodes a subunit of the spliceosome-activating Prp19 complex, which is essential for efficient pre-mRNA splicing. Loss of zygotic fas function globally impairs the efficiency of splicing, and is associated with widespread retention of introns in mRNAs and dramatic changes in gene expression. Surprisingly, despite these general effects, zygotic fas mutants show specific defects in tracheal cell migration during mid-embryogenesis when maternally supplied splicing factors have declined. We propose that tracheal branching, which relies on dynamic changes in gene expression, is particularly sensitive for efficient spliceosome function. Our results reveal an entry point to study requirements of the splicing machinery during organogenesis and provide a better understanding of disease phenotypes associated with mutations in general splicing factors.

  14. The nuclear RNA binding protein RBP33 influences mRNA and spliced leader RNA abundance in Trypanosoma brucei.

    PubMed

    Cirovic, Olivera; Trikin, Roman; Hoffmann, Anneliese; Doiron, Nicholas; Jakob, Martin; Ochsenreiter, Torsten

    2017-03-01

    RNA recognition motif (RRM) containing proteins are important regulators of gene expression in trypanosomes. Here we expand our current knowledge on the exclusively nuclear localized RRM domain containing protein RBP33 of Trypanosoma brucei. Overexpression of RBP33 leads to a quick growth arrest in G2/M in bloodstream form cells likely due to an overall mRNA- and spliced leader abundance decrease while the ribosomal RNAs remain unaffected. The recombinant RBP33 binds to poly(A) and random sequence RNA in vitro confirming its role as a RNA binding protein. Finally super-resolution microscopy detects RBP33 in small punctae throughout the nucleus and surrounding the nucleolus, however the signal is depleted inside the nucleolus.

  15. Cafeteria diet inhibits insulin clearance by reduced insulin-degrading enzyme expression and mRNA splicing.

    PubMed

    Brandimarti, P; Costa-Júnior, J M; Ferreira, S M; Protzek, A O; Santos, G J; Carneiro, E M; Boschero, A C; Rezende, L F

    2013-11-01

    Insulin clearance plays a major role in glucose homeostasis and insulin sensitivity in physiological and/or pathological conditions, such as obesity-induced type 2 diabetes as well as diet-induced obesity. The aim of the present work was to evaluate cafeteria diet-induced obesity-induced changes in insulin clearance and to explain the mechanisms underlying these possible changes. Female Swiss mice were fed either a standard chow diet (CTL) or a cafeteria diet (CAF) for 8 weeks, after which we performed glucose tolerance tests, insulin tolerance tests, insulin dynamics, and insulin clearance tests. We then isolated pancreatic islets for ex vivo glucose-stimulated insulin secretion as well as liver, gastrocnemius, visceral adipose tissue, and hypothalamus for subsequent protein analysis by western blot and determination of mRNA levels by real-time RT-PCR. The cafeteria diet induced insulin resistance, glucose intolerance, and increased insulin secretion and total insulin content. More importantly, mice that were fed a cafeteria diet demonstrated reduced insulin clearance and decay rate as well as reduced insulin-degrading enzyme (IDE) protein and mRNA levels in liver and skeletal muscle compared with the control animals. Furthermore, the cafeteria diet reduced IDE expression and alternative splicing in the liver and skeletal muscle of mice. In conclusion, a cafeteria diet impairs glucose homeostasis by reducing insulin sensitivity, but it also reduces insulin clearance by reducing IDE expression and alternative splicing in mouse liver; however, whether this mechanism contributes to the glucose intolerance or helps to ameliorate it remains unclear.

  16. HLA-A*68020103 shows an eight nucleotides deletion within intron 2 but has normal mRNA splicing and serological recognition.

    PubMed

    Balas, A; Sánchez-García, F; Bustamante, L; García-Sánchez, F; Vicario, J L

    2007-09-01

    A novel A*68020103 allele was completely characterized by sequencing in a Spanish bone marrow donor. A*68020103 has an eight nucleotides deletion at the 5'-end of intron 2, when compared with other A*6802 alleles. This alteration does not affect either its mRNA splicing process or serological detection.

  17. Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing.

    PubMed

    Defesche, J C; Schuurman, E J M; Klaaijsen, L N; Khoo, K L; Wiegman, A; Stalenhoef, A F H

    2008-06-01

    In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3'-splice donor site in exon 4 and R385R was associated with a new 5'-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay.

  18. Associations between genetic variants in mRNA splicing-related genes and risk of lung cancer: a pathway-based analysis from published GWASs

    PubMed Central

    Pan, Yongchu; Liu, Hongliang; Wang, Yanru; Kang, Xiaozheng; Liu, Zhensheng; Owzar, Kouros; Han, Younghun; Su, Li; Wei, Yongyue; Hung, Rayjean J.; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S.; Caporaso, Neil; Teresa Landi, Maria; Heinrich, Joachim; Risch, Angela; Wu, Xifeng; Ye, Yuanqing; Christiani, David C.; Amos, Christopher I.; Wei, Qingyi

    2017-01-01

    mRNA splicing is an important mechanism to regulate mRNA expression. Abnormal regulation of this process may lead to lung cancer. Here, we investigated the associations of 11,966 single-nucleotide polymorphisms (SNPs) in 206 mRNA splicing-related genes with lung cancer risk by using the summary data from six published genome-wide association studies (GWASs) of Transdisciplinary Research in Cancer of the Lung (TRICL) (12,160 cases and 16,838 controls) and another two lung cancer GWASs of Harvard University (984 cases and 970 controls) and deCODE (1,319 cases and 26,380 controls). We found that a total of 12 significant SNPs with false discovery rate (FDR) ≤0.05 were mapped to one novel gene PRPF6 and two previously reported genes (DHX16 and LSM2) that were also confirmed in this study. The six novel SNPs in PRPF6 were in high linkage disequilibrium and associated with PRPF6 mRNA expression in lymphoblastoid cells from 373 Europeans in the 1000 Genomes Project. Taken together, our studies shed new light on the role of mRNA splicing genes in the development of lung cancer. PMID:28304396

  19. Abnormal expression of mRNA, microRNA alteration and aberrant DNA methylation patterns in rectal adenocarcinoma

    PubMed Central

    Liu, Xianglong; Yuan, Xiangfei; Qin, Hai; Zhang, Xipeng

    2017-01-01

    Aim Rectal adenocarcinoma (READ) is a malignancy cancer with the high morbidity and motility worldwide. Our study aimed to explore the potential pathogenesis of READ through integrated analysis of gene expression profiling and DNA methylation data. Methods The miRNA, mRNA expression profiling and corresponding DNA methylation data were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNAs/ miRNAs/methylated regions (DEmRNA/DEmiRNAs) were identified in READ. The negatively correlation of DEmiRNA-DEmRNAs and DNA methylation-DEmRNAs were obtained. DEmRNAs expression was validated through quantitative real-time polymerase chain reaction (qRT-PCR) and microarray expression profiling analyses. Results 1192 dysregulated DEmRNAs, 27 dysregulated DEmiRNAs and 6403 aberrant methylation CpG sites were screened in READ compared to normal controls. 1987 negative interaction pairs among 27 DEmiRNAs and 668 DEmRNAs were predicted. 446 genes with aberrant methylation were annotated. Eventually, 50 DEmRNAs (39 down- and 11 up-regulated DEmRNAs) with hypermethylation, synchronously negatively targeted by DEmiRNAs, were identified through the correlation analysis among 446 genes with aberrant methylation and 668 DEmRNAs. 50 DEmRNAs were significantly enriched in cAMP signaling pathway, circadian entrainment and glutamatergic synapse. The validation results of expression levels of DEmRNAs through qRT-PCR and microarray analyses were compatible with our study. Conclusion 7 genes of SORCS1, PDZRN4, LONRF2, CNGA3, HAND2, RSPO2 and GNAO1 with hypermethylation and negatively regulation by DEmiRNAs might contribute to the tumorigenesis of READ. Our work might provide valuable foundation for the READ in mechanism elucidation, early diagnosis and therapeutic target identification. PMID:28350845

  20. Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.

    PubMed Central

    Stoffers, D A; Green, C B; Eipper, B A

    1989-01-01

    Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions. Images PMID:2911604

  1. Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

    PubMed Central

    Ansseau, Eugénie; Domire, Jacqueline S.; Wallace, Lindsay M.; Eidahl, Jocelyn O.; Guckes, Susan M.; Giesige, Carlee R.; Pyne, Nettie K.; Belayew, Alexandra; Harper, Scott Q.

    2015-01-01

    The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3’ untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons. PMID:25742305

  2. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy

    PubMed Central

    Wieben, Eric D.; Aleff, Ross A.; Tang, Xiaojia; Butz, Malinda L.; Kalari, Krishna R.; Highsmith, Edward W.; Jen, Jin; Vasmatzis, George; Patel, Sanjay V.; Maguire, Leo J.; Baratz, Keith H.; Fautsch, Michael P.

    2017-01-01

    Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events. PMID:28118661

  3. The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

    PubMed

    Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

    2016-01-01

    Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.

  4. Alternative splicing and developmental and hormonal regulation of porcine comparative gene identification-58 (CGI-58) mRNA.

    PubMed

    Li, X; Suh, Y; Kim, E; Moeller, S J; Lee, K

    2012-12-01

    The process of lipolysis is essential for regulating the catabolism of cellular fat stores. Therefore, knowledge of lipolysis contributes to improving porcine production, such as reducing back fat, enhancing lean meat, and controlling marbling. Comparative gene identification-58 (CGI-58) plays an important role in the multi-enzyme-mediated process of lipolysis. It was identified as the co-activator of adipose triglyceride lipase (ATGL), which performs the first step in breaking down triacylglycerol and generating diacylglycerol and NEFA. We cloned and sequenced the CGI-58 cDNA and deduced the AA sequences in 3 breeds of swine (Duroc, Berkshire, and Landrace). Homologies were found with the human, mouse, and chicken for the lipid droplet binding domain, the α/β hydrolase domain, and the lysophosphatidic acid acyltransferase (LPAAT) domain, which demonstrates conservation of CGI-58 across species. An alternatively spliced isoform with an exon 3 deletion was identified. Interestingly, this unique isoform contains the lipid droplet-binding domain but lacks the LPAAT domain due to an open reading frame (ORF) shift that creates a premature stop codon. Furthermore, porcine CGI-58 is expressed in multiple organs and tissues but is most predominant in adipose tissue. Porcine adipose and stromal-vascular (SV) cell fractionation reveals that CGI-58 and ATGL are highly expressed (P < 0.01) in mature adipocytes. The expressions of both CGI-58 and ATGL mRNA were found to increase (P < 0.05) at d 6 of SV cell culture, confirming their upregulation during adipogenesis and differentiation. Also, the results from in vitro cell culture showed that insulin decreased (P < 0.05) the expressions of both CGI-58 and ATGL in a dose-dependent manner. Overall, these results report the cDNA and AA sequences of porcine CGI-58 with identification of its unique alternatively spliced variant. The results of the study also reveal the developmental and hormonal regulation of porcine CGI-58 gene

  5. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  6. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.

    PubMed

    Krainer, A R; Maniatis, T; Ruskin, B; Green, M R

    1984-04-01

    Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5' or 3' end, a 3' poly A tail, or a 5'-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%-40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain beta-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.

  7. An intronic insertion in KPL2 results in aberrant splicing and causes the immotile short-tail sperm defect in the pig

    PubMed Central

    Sironen, Anu; Thomsen, Bo; Andersson, Magnus; Ahola, Virpi; Vilkki, Johanna

    2006-01-01

    The immotile short-tail sperm defect is an autosomal recessive disease within the Finnish Yorkshire pig population. This disease specifically affects the axoneme structure of sperm flagella, whereas cilia in other tissues appear unaffected. Recently, the disease locus was mapped to a 3-cM region on porcine chromosome 16. To facilitate identification of candidate genes, we constructed a porcine-human comparative map, which anchored the disease locus to a region on human chromosome 5p13.2 containing eight annotated genes. Sequence analysis of a candidate gene KPL2 revealed the presence of an inserted retrotransposon within an intron. The insertion affects splicing of the KPL2 transcript in two ways; it either causes skipping of the upstream exon, or causes the inclusion of an intronic sequence as well as part of the insertion in the transcript. Both changes alter the reading frame leading to premature termination of translation. Further work revealed that the aberrantly spliced exon is expressed predominantly in testicular tissue, which explains the tissue-specificity of the immotile short-tail sperm defect. These findings show that the KPL2 gene is important for correct axoneme development and provide insight into abnormal sperm development and infertility disorders. PMID:16549801

  8. Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

    PubMed

    Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

    2014-04-01

    Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

  9. Hibernation-specific alternative splicing of the mRNA encoding cold-inducible RNA-binding protein in the hearts of hamsters.

    PubMed

    Sano, Yuuki; Shiina, Takahiko; Naitou, Kiyotada; Nakamori, Hiroyuki; Shimizu, Yasutake

    2015-07-10

    The hearts of hibernating animals are capable of maintaining constant beating despite a decrease in body temperature to less than 10 °C during hibernation, suggesting that the hearts of hibernators are highly tolerant to a cold temperature. In the present study, we examined the expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters, since CIRP plays important roles in protection of various types of cells against harmful effects of cold temperature. RT-PCR analysis revealed that CIRP mRNA is constitutively expressed in the heart of a non-hibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP. On the other hand, the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to non-hibernating hamsters, only the short product was amplified in hibernating animals. Induction of artificial hypothermia in non-hibernating hamsters did not completely mimic the splicing patterns observed in hibernating animals, although a partial shift from long form mRNA to short form was observed. Our results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation.

  10. Nonsense-Mediated Decay of Alternative Precursor mRNA Splicing Variants Is a Major Determinant of the Arabidopsis Steady State Transcriptome[C][W

    PubMed Central

    Drechsel, Gabriele; Kahles, André; Kesarwani, Anil K.; Stauffer, Eva; Behr, Jonas; Drewe, Philipp; Rätsch, Gunnar; Wachter, Andreas

    2013-01-01

    The nonsense-mediated decay (NMD) surveillance pathway can recognize erroneous transcripts and physiological mRNAs, such as precursor mRNA alternative splicing (AS) variants. Currently, information on the global extent of coupled AS and NMD remains scarce and even absent for any plant species. To address this, we conducted transcriptome-wide splicing studies using Arabidopsis thaliana mutants in the NMD factor homologs UP FRAMESHIFT1 (UPF1) and UPF3 as well as wild-type samples treated with the translation inhibitor cycloheximide. Our analyses revealed that at least 17.4% of all multi-exon, protein-coding genes produce splicing variants that are targeted by NMD. Moreover, we provide evidence that UPF1 and UPF3 act in a translation-independent mRNA decay pathway. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. Genes generating NMD-sensitive AS variants function in diverse biological processes, including signaling and protein modification, for which NaCl stress–modulated AS-NMD was found. Besides mRNAs, numerous noncoding RNAs and transcripts derived from intergenic regions were shown to be NMD responsive. In summary, we provide evidence for a major function of AS-coupled NMD in shaping the Arabidopsis transcriptome, having fundamental implications in gene regulation and quality control of transcript processing. PMID:24163313

  11. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  12. Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

    PubMed

    Dolatshad, H; Pellagatti, A; Liberante, F G; Llorian, M; Repapi, E; Steeples, V; Roy, S; Scifo, L; Armstrong, R N; Shaw, J; Yip, B H; Killick, S; Kušec, R; Taylor, S; Mills, K I; Savage, K I; Smith, C W J; Boultwood, J

    2016-12-01

    The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

  13. Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

    PubMed Central

    Dolatshad, H; Pellagatti, A; Liberante, F G; Llorian, M; Repapi, E; Steeples, V; Roy, S; Scifo, L; Armstrong, R N; Shaw, J; Yip, B H; Killick, S; Kušec, R; Taylor, S; Mills, K I; Savage, K I; Smith, C W J; Boultwood, J

    2016-01-01

    The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS. PMID:27211273

  14. Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts

    PubMed Central

    Honorine, Romy; Mosrin-Huaman, Christine; Hervouet-Coste, Nadège; Libri, Domenico; Rahmouni, A. Rachid

    2011-01-01

    The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery. PMID:21113025

  15. BhbZIP60 from Resurrection Plant Boea hygrometrica Is an mRNA Splicing-Activated Endoplasmic Reticulum Stress Regulator Involved in Drought Tolerance

    PubMed Central

    Wang, Bo; Du, Hong; Zhang, Zhennan; Xu, Wenzhong; Deng, Xin

    2017-01-01

    Adverse environmental conditions cause endoplasmic reticulum (ER) stress in plants. To mitigate ER stress damage, ER associated transcription factors and inositol-requiring enzyme-1 (IRE1)-mediated bZIP60 mRNA splicing are activated in plants. A drought-induced gene, encoding the ortholog of AtbZIP60, was identified in the resurrection plant Boea hygrometrica, termed BhbZIP60. In response to ER stress and dehydration, BhbZIP60 mRNA can be spliced to create a frame shift in the C terminus by the excision of 23b segment in a manner of its ortholog in other plants, thus translocating to the nucleus instead of the cytoplasm. The splicing-activated BhbZIP60 (BhbZIP60S) could function in the same way as its Arabidopsis ortholog by restoring the molecular phenotype of the mutant atbzip60. When overexpressed in Arabidopsis, BhbZIP60S provided transgenic plants with enhanced tolerance to drought, tunicamycin and mannitol stresses with upregulation of the expressions of ER quality control (QC) genes (BiP2, BiP3, CNX1, and sPDI) and abscisic acid (ABA) responsive genes (RD29A, RAB18, and RD17). Furthermore, in the yeast one-hybrid system, BhbZIP60S was capable of interacting with ER stress responsive elements (ERSE and ERSE-II) that exist in the promoters of known ER-QC genes, but not binding to ABA responsive cis-elements (ABREs). Our results demonstrated that drought-induced BhbZIP60 may have a function in drought tolerance via the splicing-activated BhbZIP60S to mediate ER-QC by direct binding to the promoters of ER-QC genes. This study evidently demonstrates the involvement of ER-QC in the drought tolerance of Arabidopsis and the desiccation tolerance of the resurrection plant B. hygrometrica. PMID:28286511

  16. BhbZIP60 from Resurrection Plant Boea hygrometrica Is an mRNA Splicing-Activated Endoplasmic Reticulum Stress Regulator Involved in Drought Tolerance.

    PubMed

    Wang, Bo; Du, Hong; Zhang, Zhennan; Xu, Wenzhong; Deng, Xin

    2017-01-01

    Adverse environmental conditions cause endoplasmic reticulum (ER) stress in plants. To mitigate ER stress damage, ER associated transcription factors and inositol-requiring enzyme-1 (IRE1)-mediated bZIP60 mRNA splicing are activated in plants. A drought-induced gene, encoding the ortholog of AtbZIP60, was identified in the resurrection plant Boea hygrometrica, termed BhbZIP60. In response to ER stress and dehydration, BhbZIP60 mRNA can be spliced to create a frame shift in the C terminus by the excision of 23b segment in a manner of its ortholog in other plants, thus translocating to the nucleus instead of the cytoplasm. The splicing-activated BhbZIP60 (BhbZIP60S) could function in the same way as its Arabidopsis ortholog by restoring the molecular phenotype of the mutant atbzip60. When overexpressed in Arabidopsis, BhbZIP60S provided transgenic plants with enhanced tolerance to drought, tunicamycin and mannitol stresses with upregulation of the expressions of ER quality control (QC) genes (BiP2, BiP3, CNX1, and sPDI) and abscisic acid (ABA) responsive genes (RD29A, RAB18, and RD17). Furthermore, in the yeast one-hybrid system, BhbZIP60S was capable of interacting with ER stress responsive elements (ERSE and ERSE-II) that exist in the promoters of known ER-QC genes, but not binding to ABA responsive cis-elements (ABREs). Our results demonstrated that drought-induced BhbZIP60 may have a function in drought tolerance via the splicing-activated BhbZIP60S to mediate ER-QC by direct binding to the promoters of ER-QC genes. This study evidently demonstrates the involvement of ER-QC in the drought tolerance of Arabidopsis and the desiccation tolerance of the resurrection plant B. hygrometrica.

  17. Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

    PubMed

    Ferraldeschi, Roberta; Welti, Jonathan; Powers, Marissa V; Yuan, Wei; Smyth, Tomoko; Seed, George; Riisnaes, Ruth; Hedayat, Somaieh; Wang, Hannah; Crespo, Mateus; Nava Rodrigues, Daniel; Figueiredo, Ines; Miranda, Susana; Carreira, Suzanne; Lyons, John F; Sharp, Swee; Plymate, Stephen R; Attard, Gerhardt; Wallis, Nicola; Workman, Paul; de Bono, Johann S

    2016-05-01

    Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR.

  18. Age-Dependent Decrease and Alternative Splicing of Methionine Synthase mRNA in Human Cerebral Cortex and an Accelerated Decrease in Autism

    PubMed Central

    Muratore, Christina R.; Hodgson, Nathaniel W.; Trivedi, Malav S.; Abdolmaleky, Hamid M.; Persico, Antonio M.; Lintas, Carla; De La Monte, Suzanne; Deth, Richard C.

    2013-01-01

    The folate and vitamin B12-dependent enzyme methionine synthase (MS) is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin) from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression. PMID:23437274

  19. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

    PubMed Central

    Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  20. Recurrent disruption of the Imu splice donor site in t(14;18) positive lymphomas: a potential molecular basis for aberrant downstream class switch recombination.

    PubMed

    Ruminy, Philippe; Jardin, Fabrice; Penther, Dominique; Picquenot, Jean-Michel; Parmentier, Françoise; Buchonnet, Gérard; Bertrand, Philippe; Tilly, Hervé; Bastard, Christian

    2007-08-01

    t(14;18) positive lymphomas are mature germinal center B-cell neoplasms. In agreement with this cellular origin, most have somatically mutated immunoglobulin variable genes and the IGH@ locus has almost always been reorganized by class switch recombination (CSR). However, contrasting with normal B-cells, a majority of cases still express an IgM while the constant genes are normally rearranged only on the non-productive allele. Concurrently, aberrant intra-allelic junctions involving downstream switch regions, with a lack of engagement of the switch mu (Smu), often accumulate on the functional alleles, suggesting some recurrent CSR perturbation during the onset of the disease. To clarify these surprising observations, we addressed the accessibility of the Smu to the CSR machinery in a large series of patients by characterizing the mutations that are expected to accumulate at this place upon CSR activation. Our data indicate that the Smu is mutated in a large majority of cases, often on both alleles, indicating that these cells usually reach a differentiation stage where CSR is activated and where this region remains accessible. Interestingly, we also identified a significant cluster of mutations at the splicing donor site of the first exon of the Smu germline transcripts, on the functional allele. This location suggests a possible relation with CSR perturbations in lymphoma and the clustering points to a probable mechanism of selection. In conclusion, our data suggest that an acquired mutation at the splicing donor site of the Smu transcripts may participate in the selection of lymphoma cells and play a significant role during the onset of the disease.

  1. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells.

    PubMed Central

    Reddy, V B; Ghosh, P K; Lebowitz, P; Piatak, M; Weissman, S M

    1979-01-01

    We have studied the structure of polyadenylated virus-specific cytoplasmic mRNA's in mouse and human cells transformed by simian virus 40 and in monkey cells infected with simian virus 40 in the presence of cytosine arabinoside by means of reverse transcriptase-catalyzed complementary DNA synthesis and complementary DNA sequencing. Abundant mRNA species containing splices from residues 4490 to 4557 (0.533 to 0.546 map units [m.u.]) and 4490 to 4837 (0.533 to 0.600 m.u.) were identified in both transformed and infected cells. Two principal reverse transcriptase stops were observed at the 5' termini of these mRNA's, both occurring with approximately equal frequency. The most distal of these stops was localized at residues 5152 to 5154 (0.660 m.u.), and the second was at residues 5147 to 5148 (0.659 m.u.). Several additional minor stops, between approximately 0.62 and 0.65 m.u., were also found on complementary DNA copied from transformed cell mRNA; in contrast, only one additional stop was present on complementary DNA copied from early lytic mRNA. These data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional 5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's. Transformed cell mRNA was also found to contain a single 3' terminus at positions 2504 and 2505 (0.153 m.u.); termini lying beyond this site were not detected. Images PMID:90157

  2. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

    PubMed Central

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  3. A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing.

    PubMed

    Wilmes, Gwendolyn M; Bergkessel, Megan; Bandyopadhyay, Sourav; Shales, Michael; Braberg, Hannes; Cagney, Gerard; Collins, Sean R; Whitworth, Gregg B; Kress, Tracy L; Weissman, Jonathan S; Ideker, Trey; Guthrie, Christine; Krogan, Nevan J

    2008-12-05

    We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA-processing pathways. Due to their complexity and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here, we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.

  4. Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

    PubMed

    Snijders, Ambrosius P; Hautbergue, Guillaume M; Bloom, Alex; Williamson, James C; Minshull, Thomas C; Phillips, Helen L; Mihaylov, Simeon R; Gjerde, Douglas T; Hornby, David P; Wilson, Stuart A; Hurd, Paul J; Dickman, Mark J

    2015-03-01

    Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

  5. Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation

    PubMed Central

    Breig, Osman; Baklouti, Faouzi

    2013-01-01

    SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development. PMID:23536862

  6. Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing.

    PubMed

    van Kuilenburg, André B P; Meijer, Judith; Maurer, Dirk; Dobritzsch, Doreen; Meinsma, Rutger; Los, Maartje; Knegt, Lia C; Zoetekouw, Lida; Jansen, Rob L H; Dezentjé, Vincent; van Huis-Tanja, Lieke H; van Kampen, Roel J W; Hertz, Jens Michael; Hennekam, Raoul C M

    2017-03-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

  7. Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome.

    PubMed

    Colla, Simona; Ong, Derrick Sek Tong; Ogoti, Yamini; Marchesini, Matteo; Mistry, Nipun A; Clise-Dwyer, Karen; Ang, Sonny A; Storti, Paola; Viale, Andrea; Giuliani, Nicola; Ruisaard, Kathryn; Ganan Gomez, Irene; Bristow, Christopher A; Estecio, Marcos; Weksberg, David C; Ho, Yan Wing; Hu, Baoli; Genovese, Giannicola; Pettazzoni, Piergiorgio; Multani, Asha S; Jiang, Shan; Hua, Sujun; Ryan, Michael C; Carugo, Alessandro; Nezi, Luigi; Wei, Yue; Yang, Hui; D'Anca, Marianna; Zhang, Li; Gaddis, Sarah; Gong, Ting; Horner, James W; Heffernan, Timothy P; Jones, Philip; Cooper, Laurence J N; Liang, Han; Kantarjian, Hagop; Wang, Y Alan; Chin, Lynda; Bueso-Ramos, Carlos; Garcia-Manero, Guillermo; DePinho, Ronald A

    2015-05-11

    Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

  8. BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

    PubMed Central

    Liu, Wenbo; Wang, Fengchao; Xu, Qianhua; Shi, Junchao; Zhang, Xiaoxin; Lu, Xukun; Zhao, Zhen-Ao; Gao, Zheng; Ma, Huaixiao; Duan, Enkui; Gao, Fei; Gao, Shaorong; Yi, Zhaohong; Li, Lei

    2017-01-01

    Breast cancer amplified sequence 2 (BCAS2) is involved in multiple biological processes, including pre-mRNA splicing. However, the physiological roles of BCAS2 are still largely unclear. Here we report that BCAS2 is specifically enriched in spermatogonia of mouse testes. Conditional disruption of Bcas2 in male germ cells impairs spermatogenesis and leads to male mouse infertility. Although the spermatogonia appear grossly normal, spermatocytes in meiosis prophase I and meiosis events (recombination and synapsis) are rarely observed in the BCAS2-depleted testis. In BCAS2 null testis, 245 genes are altered in alternative splicing forms; at least three spermatogenesis-related genes (Dazl, Ehmt2 and Hmga1) can be verified. In addition, disruption of Bcas2 results in a significant decrease of the full-length form and an increase of the short form (lacking exon 8) of DAZL protein. Altogether, our results suggest that BCAS2 regulates alternative splicing in spermatogonia and the transition to meiosis initiation, and male fertility. PMID:28128212

  9. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  10. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  11. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells.

    PubMed

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-09-01

    Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10-15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy.

  12. Aberrant hypomethylated STAT3 was identified as a biomarker of chronic benzene poisoning through integrating DNA methylation and mRNA expression data.

    PubMed

    Yang, Jing; Bai, Wenlin; Niu, Piye; Tian, Lin; Gao, Ai

    2014-06-01

    Chronic occupational benzene exposure is associated with an increased risk of hematological malignancies such as aplastic anemia and leukemia. The new biomarker and action mechanisms of chronic benzene poisoning are still required to be explored. Aberrant DNA methylation, which may lead to genomic instability and the altered gene expression, is frequently observed in hematological cancers. To gain an insight into the new biomarkers and molecular mechanisms of chronic benzene poisoning, DNA methylation profiles and mRNA expression pattern from the peripheral blood mononuclear cells of four chronic benzene poisoning patients and four health controls that matched age and gender without benzene exposure were performed using the high resolution Infinium 450K methylation array and Gene Chip Human Gene 2.0ST Arrays, respectively. By integrating DNA methylation and mRNA expression data, we identified 3 hypermethylated genes showing concurrent down-regulation (PRKG1, PARD3, EPHA8) and 2 hypomethylated genes showing increased expression (STAT3, IFNGR1). Signal net analysis of differential methylation genes associated with chronic benzene poisoning showed that two key hypomethylated STAT3 and hypermethylated GNAI1 were identified. Further GO analysis and pathway analysis indicated that hypomethylated STAT3 played central roles through regulation of transcription, DNA-dependent, positive regulation of transcription from RNA polymerase II promoter, JAK-STAT cascade and adipocytokine signaling pathway, Acute myeloid leukemia, and JAK-STAT signaling pathway. In conclusion, the aberrant hypomethylated STAT3 might be a potential biomarker of chronic benzene poisoning.

  13. ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing.

    PubMed

    Gatto, Sole; Gagliardi, Miriam; Franzese, Monica; Leppert, Sylwia; Papa, Mariarosaria; Cammisa, Marco; Grillo, Giacomo; Velasco, Guillame; Francastel, Claire; Toubiana, Shir; D'Esposito, Maurizio; Angelini, Claudia; Matarazzo, Maria R

    2017-03-09

    Hypomorphic mutations in DNA-methyltransferase DNMT3B cause majority of the rare disorder Immunodeficiency, Centromere instability and Facial anomalies syndrome cases (ICF1). By unspecified mechanisms, mutant-DNMT3B interferes with lymphoid-specific pathways resulting in immune response defects. Interestingly, recent findings report that DNMT3B shapes intragenic CpG-methylation of highly-transcribed genes. However, how the DNMT3B-dependent epigenetic network modulates transcription and whether ICF1-specific mutations impair this process remains unknown. We performed a transcriptomic and epigenomic study in patient-derived B-cell lines to investigate the genome-scale effects of DNMT3B dysfunction. We highlighted that altered intragenic CpG-methylation impairs multiple aspects of transcriptional regulation, like alternative TSS usage, antisense transcription and exon splicing. These defects preferentially associate with changes of intragenic H3K4me3 and at lesser extent of H3K27me3 and H3K36me3. In addition, we highlighted a novel DNMT3B activity in modulating the self-regulatory circuit of sense-antisense pairs and the exon skipping during alternative splicing, through interacting with RNA molecules. Strikingly, altered transcription affects disease relevant genes, as for instance the memory-B cell marker CD27 and PTPRC genes, providing us with biological insights into the ICF1-syndrome pathogenesis. Our genome-scale approach sheds light on the mechanisms still poorly understood of the intragenic function of DNMT3B and DNA methylation in gene expression regulation.

  14. Functional Genomic mRNA Profiling of Colorectal Adenomas: Identification and in vivo Validation of CD44 and Splice Variant CD44v6 as Molecular Imaging Targets.

    PubMed

    Hartmans, Elmire; Orian-Rousseau, Veronique; Matzke-Ogi, Alexandra; Karrenbeld, Arend; de Groot, Derk Jan A; de Jong, Steven; van Dam, Gooitzen M; Fehrmann, Rudolf S N; Nagengast, Wouter B

    2017-01-01

    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. High adenoma miss rates, especially seen in high-risk patients, demand for better endoscopic detection. By fluorescently 'highlighting' specific molecular characteristics, endoscopic molecular imaging has great potential to fulfill this need. To implement this technique effectively, target proteins that distinguish adenomas from normal tissue must be identified. In this study we applied in silico Functional Genomic mRNA (FGmRNA) profiling, which is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations occurring in adenomas on gene expression levels. FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to identify overexpressed genes. We validated the protein expression of the top identified genes, AXIN2, CEMIP, CD44 and JUN, in sporadic adenoma patient samples via immunohistochemistry (IHC). CD44 was identified as the most attractive target protein for imaging purposes and we proved its relevance in high-risk patients by demonstrating CD44 protein overexpression in Lynch lesions. Subsequently, we show that the epithelial splice variant CD44V6 is highly overexpressed in our patient samples and we demonstrated the feasibility of visualizing adenomas in Apc(Min/+) mice in vivo by using a fluorescently labeled CD44v6 targeting peptide. In conclusion, via in silico functional genomics and ex vivo protein validation, this study identified CD44 as an attractive molecular target for both sporadic and high-risk Lynch adenomas, and demonstrates the in vivo applicability of a small peptide drug directed against splice variant CD44v6 for adenoma imaging.

  15. Functional Genomic mRNA Profiling of Colorectal Adenomas: Identification and in vivo Validation of CD44 and Splice Variant CD44v6 as Molecular Imaging Targets

    PubMed Central

    Hartmans, Elmire; Orian-Rousseau, Veronique; Matzke-Ogi, Alexandra; Karrenbeld, Arend; de Groot, Derk Jan A.; de Jong, Steven; van Dam, Gooitzen M.; Fehrmann, Rudolf S.N.; Nagengast, Wouter B.

    2017-01-01

    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. High adenoma miss rates, especially seen in high-risk patients, demand for better endoscopic detection. By fluorescently 'highlighting' specific molecular characteristics, endoscopic molecular imaging has great potential to fulfill this need. To implement this technique effectively, target proteins that distinguish adenomas from normal tissue must be identified. In this study we applied in silico Functional Genomic mRNA (FGmRNA) profiling, which is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations occurring in adenomas on gene expression levels. FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to identify overexpressed genes. We validated the protein expression of the top identified genes, AXIN2, CEMIP, CD44 and JUN, in sporadic adenoma patient samples via immunohistochemistry (IHC). CD44 was identified as the most attractive target protein for imaging purposes and we proved its relevance in high-risk patients by demonstrating CD44 protein overexpression in Lynch lesions. Subsequently, we show that the epithelial splice variant CD44V6 is highly overexpressed in our patient samples and we demonstrated the feasibility of visualizing adenomas in ApcMin/+ mice in vivo by using a fluorescently labeled CD44v6 targeting peptide. In conclusion, via in silico functional genomics and ex vivo protein validation, this study identified CD44 as an attractive molecular target for both sporadic and high-risk Lynch adenomas, and demonstrates the in vivo applicability of a small peptide drug directed against splice variant CD44v6 for adenoma imaging. PMID:28255344

  16. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  17. Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells

    PubMed Central

    Hatakeyama, Shingo; Sugihara, Kazuhiro; Nakayama, Jun; Akama, Tomoya O.; Wong, Shuk-Man Annie; Kawashima, Hiroto; Zhang, Jianing; Smith, David F.; Ohyama, Chikara; Fukuda, Minoru; Fukuda, Michiko N.

    2009-01-01

    Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1, Sfrs2, Sfrs5, and Sfrs7 gene products. Bacterially expressed Sfrs1 protein bound to B16-FTIII-M cells but not to parental B16 cells. Recombinant Sfrs1 protein bound to a series of fucosylated oligosaccharides in glycan array and plate-binding assays. When anti-Sfrs antibodies were injected intravenously into mice, antibodies labeled a subset of lung capillaries. Anti-Sfrs antibodies inhibited homing of I-peptide-displaying phage to the lung colonization of B16-FTIII-M cells in vivo in the mouse. These results strongly suggest that Sfrs proteins are responsible for fucosylated carbohydrate-dependent lung metastasis of epithelial cancers. PMID:19218444

  18. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.

    PubMed

    Ono, Ryuichi; Ishii, Masayuki; Fujihara, Yoshitaka; Kitazawa, Moe; Usami, Takako; Kaneko-Ishino, Tomoko; Kanno, Jun; Ikawa, Masahito; Ishino, Fumitoshi

    2015-07-28

    The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

  19. Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis

    PubMed Central

    Nevarez, Lisette; Pogue, Robert; Krakow, Deborah; Cohn, Daniel H.

    2016-01-01

    The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses. PMID:27622494

  20. Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

    PubMed

    Souwer, Yuri; Chamuleau, Martine E D; van de Loosdrecht, Arjan A; Tolosa, Eva; Jorritsma, Tineke; Muris, Jettie J F; Dinnissen-van Poppel, Marion J; Snel, Sander N; van de Corput, Lisette; Ossenkoppele, Gert J; Meijer, Chris J L M; Neefjes, Jacques J; Marieke van Ham, S

    2009-05-01

    In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

  1. Minor class splicing shapes the zebrafish transcriptome during development.

    PubMed

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M; Doggett, Karen; Keightley, Maria-Cristina; Boglev, Yeliz; Trotter, Andrew J; Ng, Annie Y; Wilkins, Simon J; Verkade, Heather; Ober, Elke A; Field, Holly A; Grimmond, Sean M; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2014-02-25

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

  2. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43.

    PubMed

    Arnold, Eveline S; Ling, Shuo-Chien; Huelga, Stephanie C; Lagier-Tourenne, Clotilde; Polymenidou, Magdalini; Ditsworth, Dara; Kordasiewicz, Holly B; McAlonis-Downes, Melissa; Platoshyn, Oleksandr; Parone, Philippe A; Da Cruz, Sandrine; Clutario, Kevin M; Swing, Debbie; Tessarollo, Lino; Marsala, Martin; Shaw, Christopher E; Yeo, Gene W; Cleveland, Don W

    2013-02-19

    Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.

  3. Hodgkin's lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1.

    PubMed

    Kato, Masato; Khan, Seema; Gonzalez, Nelson; O'Neill, Brian P; McDonald, Kylie J; Cooper, Ben J; Angel, Nicola Z; Hart, Derek N J

    2003-09-05

    Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

  4. The Intronic GABRG2 Mutation, IVS6+2T→G, Associated with CAE Altered Subunit mRNA Intron Splicing, Activated Nonsense-Mediated Decay and Produced a Stable Truncated γ2 Subunit

    PubMed Central

    Tian, Mengnan; Macdonald, Robert L.

    2012-01-01

    The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy (CAE) and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wildtype and mutant γ2S subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wildtype and mutant γ2S subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wildtype and mutant GABRG2 mRNA splicing patterns were determined in both BAC transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention and produced a frame shift in exon 7 that created a premature translation-termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense mediated mRNA decay (NMD) or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first 6 GABRG2 exons and a novel frame-shifted 29 aa C terminal tail. The γ2-PTC subunit was homologous to the mollusk acetylcholine binding protein (AChBP) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant negative effect on αβγ2 receptor assembly. PMID:22539854

  5. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

    PubMed Central

    Arnold, Eveline S.; Ling, Shuo-Chien; Huelga, Stephanie C.; Lagier-Tourenne, Clotilde; Polymenidou, Magdalini; Ditsworth, Dara; Kordasiewicz, Holly B.; McAlonis-Downes, Melissa; Platoshyn, Oleksandr; Parone, Philippe A.; Da Cruz, Sandrine; Clutario, Kevin M.; Swing, Debbie; Tessarollo, Lino; Marsala, Martin; Shaw, Christopher E.; Yeo, Gene W.; Cleveland, Don W.

    2013-01-01

    Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43Q331K and TDP-43M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage. PMID:23382207

  6. [Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

    PubMed

    Pepe, Carolina M; Saraco, Nora I; Baquedano, María Sonia; Guercio, Gabriela; Vaiani, Elisa; Berensztein, Esperanza; Rivarola, Marco A; Belgorosky, Alicia

    2007-01-01

    P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

  7. Alternative RNA splicing and cancer.

    PubMed

    Liu, Sali; Cheng, Chonghui

    2013-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells.

  8. Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

    PubMed

    Faà, Valeria; Incani, Federica; Meloni, Alessandra; Corda, Denise; Masala, Maddalena; Baffico, A Maria; Seia, Manuela; Cao, Antonio; Rosatelli, M Cristina

    2009-10-30

    Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

  9. Alternative splicing interference by xenobiotics.

    PubMed

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

  10. Targeting RNA Splicing for Disease Therapy

    PubMed Central

    Havens, Mallory A.; Duelli, Dominik M.

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics. PMID:23512601

  11. Targeting RNA splicing for disease therapy.

    PubMed

    Havens, Mallory A; Duelli, Dominik M; Hastings, Michelle L

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics.

  12. Effects of Age and Hindlimb Immobilization and Remobilization on Fast Troponin T Precursor mRNA Alternative Splicing in Rat Gastrocnemius Muscle

    PubMed Central

    Ravi, Suhana; Schilder, Rudolf J.; Berg, Arthur S.; Kimball, Scot R.

    2016-01-01

    Fast skeletal muscle Troponin T (TNNT3) is an important component of the skeletal muscle contractile machinery. The pre-mRNA encoding TNNT3 is alternatively spliced and changes in the pattern of TNNT3 splice form expression are associated with alterations in thin filament calcium sensitivity and force production during muscle contraction, thereby regulating muscle function. Interestingly, during aging, muscle force/cross sectional area is reduced, suggesting that loss of mass does not completely account for the impaired muscle function that develops during the aging process. Therefore, in the present study, we tested the hypothesis that age- and changes in muscle loading are associated with alterations in TNNT3 alternative splicing in the rat gastrocnemius muscle. We found that the relative abundance of several TNNT3 splice forms varied significantly with age among 2, 9, and 18-month old rats, and the pattern correlated with changes in body weight rather than muscle mass. Hindlimb immobilization for 7 days resulted in dramatic alterations in splice form relative abundance such that the pattern was similar to that observed in lighter animals. Remobilization for 7 days restored the splicing pattern toward that observed in the non-immobilized limb, even though muscle mass had not yet begun to recover. In conclusion, the results suggest that TNNT3 pre-mRNA alternative splicing is rapidly (i.e. within days) modulated in response to changes in the load placed on the muscle. Moreover, the results show that restoration of TNNT3 alternative splicing to control patterns is initiated prior to an increase in muscle mass. PMID:26799695

  13. N1303K (c.3909C>G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C>T] Complex Allele in CFTR Exon 7 Aberrant Splicing

    PubMed Central

    Farhat, Raëd; Puissesseau, Géraldine; El-Seedy, Ayman; Pasquet, Marie-Claude; Adolphe, Catherine; Corbani, Sandra; Megarbané, André; Kitzis, Alain; Ladeveze, Véronique

    2015-01-01

    Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasians. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys, old nomenclature: N1303K) is one of the most common worldwide mutations. This mutation has been found at high frequencies in the Mediterranean countries with the highest frequency in the Lebanese population. Therefore, on the genetic level, we conducted a complete CFTR gene screening on c.3909C>G Lebanese patients. The complex allele c.[744-33GATT(6); 869+11C>T] was always associated with the c.3909C>G mutation in cis in the Lebanese population. In cellulo splicing studies, realized by hybrid minigene constructs, revealed no impact of the c.3909C>G mutation on the splicing process, whereas the associated complex allele induces minor exon skipping. PMID:26075213

  14. RNA splicing: disease and therapy.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2011-05-01

    The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.

  15. Interaction of the Epstein-Barr virus mRNA export factor EB2 with human Spen proteins SHARP, OTT1, and a novel member of the family, OTT3, links Spen proteins with splicing regulation and mRNA export.

    PubMed

    Hiriart, Edwige; Gruffat, Henri; Buisson, Monique; Mikaelian, Ivan; Keppler, Selina; Meresse, Patrick; Mercher, Thomas; Bernard, Olivier A; Sergeant, Alain; Manet, Evelyne

    2005-11-04

    The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), a protein absolutely required for the production of infectious virions, shares properties with mRNA export factors. By using a yeast two-hybrid screen, we have identified the human protein OTT3 as an EB2-interacting factor. OTT3 is a new member of the Spen (split end) family of proteins (huSHARP, huOTT1, DmSpen, and muMINT), which are characterized by several N-terminal RNA recognition motifs and a highly conserved C-terminal SPOC (Spen Paralog and Ortholog C-terminal) domain that, in the case of SHARP, has been shown to interact with SMRT/NCoR corepressors. OTT3 is ubiquitously expressed as a 120-kDa protein. Transfected OTT3 is a nonshuttling nuclear protein that co-localizes with co-transfected EB2. We also showed that EB2 interacts with the SPOC domains of both OTT1 and SHARP proteins. Although the OTT3 interaction domain maps within the 40 N-terminal amino acids of EB2, OTT1 and SHARP interact within the C-terminal half of the protein. Furthermore, we demonstrated that the capacity of the OTT3 and OTT1 SPOC domains to interact with SMRT and repress transcription is far weaker than that of SHARP. Thus there is no evidence for a role of OTT3 in transcriptional regulation. Most interestingly, however, we have found that OTT3 has a role in splicing regulation; OTT3 represses accumulation of the alternatively spliced beta-thalassemia mRNAs, but it has no effect on the beta-globin constitutively spliced mRNA. Thus our results suggested a new function for Spen proteins related to mRNA export and splicing.

  16. Alternatively spliced T-cell receptor transcripts are up-regulated in response to disruption of either splicing elements or reading frame.

    PubMed

    Chang, Yao-Fu; Chan, Wai-Kin; Imam, J Saadi; Wilkinson, Miles F

    2007-10-12

    Nonsense mutations create premature termination codons (PTCs), leading to the generation of truncated proteins, some of which have deleterious gain-of-function or dominant-negative activity. Protecting cells from such aberrant proteins is non-sense-mediated decay (NMD), an RNA surveillance pathway that degrades transcripts harboring PTCs. A second response to nonsense mutations is the up-regulation of alternatively spliced transcripts that skip the PTC. This nonsense-associated altered splicing (NAS) response has the potential to rescue protein function, but the mechanism by which it is triggered has been controversial. Some studies suggest that, like NMD, NAS is triggered as a result of nonsense mutations disrupting reading frame, whereas other studies suggest that NAS is triggered when nonsense mutations disrupt exonic splicing enhancers (ESEs). Using T-cell receptor-beta (TCRbeta), which naturally acquires PTCs at high frequency, we provide evidence that both mechanisms act on a single type of mRNA. Mutations that disrupt consensus ESE sites up-regulated an alternatively spliced TCRbeta transcript that skipped the mutations independently of reading frame disruption and the NMD factor UPF1. In contrast, reading frame-disrupting mutations that did not disrupt consensus ESE sites elicited UPF1-dependent up-regulation of the alternatively spliced TCRbeta transcript. Restoration of reading frame prevented this up-regulation. Our results suggest that the response of an mRNA to a nonsense mutation depends on its context.

  17. An Aberrant Splice Acceptor Site Due to a Novel Intronic Nucleotide Substitution in MSX1 Gene Is the Cause of Congenital Tooth Agenesis in a Japanese Family

    PubMed Central

    Tatematsu, Tadashi; Kimura, Masashi; Nakashima, Mitsuko; Machida, Junichiro; Yamaguchi, Seishi; Shibata, Akio; Goto, Hiroki; Nakayama, Atsuo; Higashi, Yujiro; Miyachi, Hitoshi; Shimozato, Kazuo; Matsumoto, Naomichi; Tokita, Yoshihito

    2015-01-01

    Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency. PMID:26030286

  18. Alcoholism and alternative splicing of candidate genes.

    PubMed

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  19. p54nrb/NONO regulates cyclic AMP-dependent glucocorticoid production by modulating phosphodiesterase mRNA splicing and degradation.

    PubMed

    Lu, Jia Yang; Sewer, Marion B

    2015-04-01

    Glucocorticoid production in the adrenal cortex is activated in response to an increase in cyclic AMP (cAMP) signaling. The nuclear protein p54(nrb)/NONO belongs to the Drosophila behavior/human splicing (DBHS) family and has been implicated in several nuclear processes, including transcription, splicing, and RNA export. We previously identified p54(nrb)/NONO as a component of a protein complex that regulates the transcription of CYP17A1, a gene required for glucocorticoid production. Based on the multiple mechanisms by which p54(nrb)/NONO has been shown to control gene expression and the ability of the protein to be recruited to the CYP17A1 promoter, we sought to further define the molecular mechanism by which p54(nrb)/NONO confers optimal cortisol production. We show here that silencing p54(nrb)/NONO expression in H295R human adrenocortical cells decreases the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to adrenocorticotropin hormone (ACTH) stimulation. Interestingly, the expression of multiple phosphodiesterase (PDE) isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54(nrb)/NONO knockdown cells. Investigation of the mechanism by which silencing of p54(nrb)/NONO led to increased expression of select PDE isoforms revealed that p54(nrb)/NONO regulates the splicing of a subset of PDE isoforms. Importantly, we also identify a role for p54(nrb)/NONO in regulating the stability of PDE transcripts by facilitating the interaction between the exoribonuclease XRN2 and select PDE transcripts. In summary, we report that p54(nrb)/NONO modulates cAMP-dependent signaling, and ultimately cAMP-stimulated glucocorticoid biosynthesis by regulating the splicing and degradation of PDE transcripts.

  20. p54nrb/NONO Regulates Cyclic AMP-Dependent Glucocorticoid Production by Modulating Phosphodiesterase mRNA Splicing and Degradation

    PubMed Central

    Lu, Jia Yang

    2015-01-01

    Glucocorticoid production in the adrenal cortex is activated in response to an increase in cyclic AMP (cAMP) signaling. The nuclear protein p54nrb/NONO belongs to the Drosophila behavior/human splicing (DBHS) family and has been implicated in several nuclear processes, including transcription, splicing, and RNA export. We previously identified p54nrb/NONO as a component of a protein complex that regulates the transcription of CYP17A1, a gene required for glucocorticoid production. Based on the multiple mechanisms by which p54nrb/NONO has been shown to control gene expression and the ability of the protein to be recruited to the CYP17A1 promoter, we sought to further define the molecular mechanism by which p54nrb/NONO confers optimal cortisol production. We show here that silencing p54nrb/NONO expression in H295R human adrenocortical cells decreases the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to adrenocorticotropin hormone (ACTH) stimulation. Interestingly, the expression of multiple phosphodiesterase (PDE) isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54nrb/NONO knockdown cells. Investigation of the mechanism by which silencing of p54nrb/NONO led to increased expression of select PDE isoforms revealed that p54nrb/NONO regulates the splicing of a subset of PDE isoforms. Importantly, we also identify a role for p54nrb/NONO in regulating the stability of PDE transcripts by facilitating the interaction between the exoribonuclease XRN2 and select PDE transcripts. In summary, we report that p54nrb/NONO modulates cAMP-dependent signaling, and ultimately cAMP-stimulated glucocorticoid biosynthesis by regulating the splicing and degradation of PDE transcripts. PMID:25605330

  1. Benchmark analysis of algorithms for determining and quantifying full-length mRNA splice forms from RNA-seq data

    PubMed Central

    Hayer, Katharina E.; Pizarro, Angel; Lahens, Nicholas F.; Hogenesch, John B.; Grant, Gregory R.

    2015-01-01

    Motivation: Because of the advantages of RNA sequencing (RNA-Seq) over microarrays, it is gaining widespread popularity for highly parallel gene expression analysis. For example, RNA-Seq is expected to be able to provide accurate identification and quantification of full-length splice forms. A number of informatics packages have been developed for this purpose, but short reads make it a difficult problem in principle. Sequencing error and polymorphisms add further complications. It has become necessary to perform studies to determine which algorithms perform best and which if any algorithms perform adequately. However, there is a dearth of independent and unbiased benchmarking studies. Here we take an approach using both simulated and experimental benchmark data to evaluate their accuracy. Results: We conclude that most methods are inaccurate even using idealized data, and that no method is highly accurate once multiple splice forms, polymorphisms, intron signal, sequencing errors, alignment errors, annotation errors and other complicating factors are present. These results point to the pressing need for further algorithm development. Availability and implementation: Simulated datasets and other supporting information can be found at http://bioinf.itmat.upenn.edu/BEERS/bp2 Supplementary information: Supplementary data are available at Bioinformatics online. Contact: hayer@upenn.edu PMID:26338770

  2. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  3. Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African-American Prostate Cancer

    DTIC Science & Technology

    2015-10-01

    RNA and annotated. In addition, we have developed SSOs to manipulate PIK3CD alternative splicing, to correct aberrant splicing leading to production...molecular mechanisms, differential gene expression, alternative RNA splicing, epigenetic alterations, clinical tumor aggressiveness 16. SECURITY...words): Prostate cancer, health disparities among racial groups, molecular mechanisms, differential gene expression, alternative RNA splicing

  4. RNA splicing and splicing regulator changes in prostate cancer pathology.

    PubMed

    Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

    2017-04-05

    Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

  5. Molecular Characteristics, mRNA Expression, and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  6. The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.

    PubMed

    Zraly, Claudia B; Dingwall, Andrew K

    2012-07-01

    Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

  7. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen).

    PubMed

    Zhang, Jianzhen; Liu, Xiaojian; Zhang, Jianqin; Li, Daqi; Sun, Yi; Guo, Yaping; Ma, Enbo; Zhu, Kun Yan

    2010-11-01

    Chitin synthases are crucial enzymes responsible for chitin biosynthesis in fungi, nematodes and arthropods. We characterized two alternative splicing-derived variants of chitin synthase 1 gene (LmCHS1) from the oriental migratory locust, Locusta migratoria manilensis (Meyen). Each cDNA of the two variants (LmCHS1A and LmCHS1B) consists of 5116 nucleotides that include a 4728-nucleotide open reading frame (ORF) encoding 1576 amino acid residues, and 67- and 321-bp non-coding regions at the 5'- and 3'-ends of the cDNA, respectively. The two variants differ only in one exon consisting of 177 nucleotides that encode 59 amino acid residues. The amino acid sequences within this alternative splicing region are 75% identical between the two variants. Both variants were expressed in all the developmental stages. However, LmCHS1A was predominately expressed in the integument whereas LmCHS1B was mainly expressed in the trachea. Our RNAi-based gene silencing study resulted in a dramatic reduction in the levels of the corresponding mRNA in the locust nymphs injected with dsRNA of LmCHS1, or either of its two variants, LmCHS1A and LmCHS1B. Consequentially, 95, 88 and 51% of mortalities were observed in the locusts injected with the LmCHS1, LmCHS1A and LmCHS1B dsRNA, respectively. The phenotypes resulted from the injection of LmCHS1A dsRNA were similar to those from the injection of LmCHS1 dsRNA, whereas the locusts injected with LmCHS1B dsRNA exhibited crimpled cuticle phenotype. Our results suggest that both variants of chitin synthase 1 are essential for insect growth and development.

  8. Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

    SciTech Connect

    Cizdziel, P.E.; De Mars, M.; Murphy, E.C. Jr.

    1988-04-01

    The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33/sup 0/C or lower than at 37 to 41/sup 0/C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41/sup 0/C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. The authors exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39/sup 0/C. However, after a short (about 30-min) lag following a shift to 33/sup 0/C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA.

  9. Myotubular myopathy caused by multiple abnormal splicing variants in the MTM1 RNA in a patient with a mild phenotype

    PubMed Central

    Vasli, Nasim; Laugel, Vincent; Böhm, Johann; Lannes, Béatrice; Biancalana, Valérie; Laporte, Jocelyn

    2012-01-01

    Mutations impacting on the splicing of pre-mRNA are one important cause of genetically inherited diseases. However, detection of splice mutations, that are mainly due to intronic variations, and characterization of their effects are usually not performed as a first approach during genetic diagnosis. X-linked recessive myotubular myopathy is a severe congenital myopathy due to mutations in the MTM1 gene encoding myotubularin. Here, we screened a male patient showing an unusually mild phenotype without respiratory distress by western blot with specific myotubularin antibodies and detected a strong reduction of the protein level.The disease was subsequently linked to a hemizygous point mutation affecting the acceptor splice site of exon 8 of MTM1, proven by protein, transcript and genomic DNA analysis. Detailed analysis of the MTM1 mRNA by RT-PCR, sequencing and quantitative PCR revealed multiple abnormal transcripts with retention of a truncated exon 8, and neighboring exons 7 and 9 but exclusion of several other exons, suggesting a complex effect of this mutation on the splicing of non-adjacent exons. We conclude that the analysis of RNA by RT-PCR and sequencing is an important step to characterize the precise impact of detected splice variants. It is likely that complex splice aberrations due to a single mutation also account for unsolved cases in other diseases. PMID:22258523

  10. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.

  11. Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5′ splice site

    PubMed Central

    Yadegari, Hamideh; Biswas, Arijit; Akhter, Mohammad Suhail; Driesen, Julia; Ivaskevicius, Vytautas; Marquardt, Natascha

    2016-01-01

    Disease-associated silent mutations are considered to affect the accurate pre–messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5′ splice site (5′ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5′ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5′ss. This hairpin sequesters the 5′ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA. PMID:27543438

  12. Interleukin 7 up-regulates CD95 protein on CD4+ T cells by affecting mRNA alternative splicing: priming for a synergistic effect on HIV-1 reservoir maintenance.

    PubMed

    Yin, Yue; Zhang, Shaoying; Luo, Haihua; Zhang, Xu; Geng, Guannan; Li, Jun; Guo, Xuemin; Cai, Weiping; Li, Linghua; Liu, Chao; Zhang, Hui

    2015-01-02

    Interleukin-7 (IL-7) has been used as an immunoregulatory and latency-reversing agent in human immunodeficiency virus type 1 (HIV-1) infection. Although IL-7 can restore circulating CD4(+) T cell counts in HIV-1-infected patients, the anti-apoptotic and proliferative effects of IL-7 appear to benefit survival and expansion of HIV-1-latently infected memory CD4(+) T lymphocytes. IL-7 has been shown to elevate CD95 on CD4(+) T cells in HIV-1-infected individuals and prime CD4(+) T lymphocytes to CD95-mediated proliferative or apoptotic signals. Here we observed that through increasing microRNA-124, IL-7 down-regulates the splicing regulator polypyrimidine tract binding protein (PTB), leading to inclusion of the transmembrane domain-encoding exon 6 of CD95 mRNA and, subsequently, elevation of CD95 on memory CD4(+) T cells. Moreover, IL-7 up-regulates cellular FLICE-like inhibitory protein (c-FLIP) and stimulates c-Jun N-terminal kinase (JNK) phosphorylation, which switches CD95 signaling to survival mode in memory CD4(+) T lymphocytes. As a result, co-stimulation through IL-7/IL-7R and FasL/CD95 signal pathways augments IL-7-mediated survival and expansion of HIV-1-latently infected memory CD4(+) T lymphocytes. Collectively, we have demonstrated a novel mechanism for IL-7-mediated maintenance of HIV-1 reservoir.

  13. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    PubMed

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  14. Methylxanthines Increase Expression of the Splicing Factor SRSF2 by Regulating Multiple Post-transcriptional Mechanisms*

    PubMed Central

    Shi, Jia; Pabon, Kirk; Scotto, Kathleen W.

    2015-01-01

    We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative autoregulatory loop. In the present study we have investigated the mechanisms by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3′ UTR splice variants. Although some of these variants were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, down-regulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeine-induced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2-targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine down-regulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeine-mediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated in disease. PMID:25818199

  15. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  16. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

  17. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

    PubMed

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

    2016-01-12

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  18. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

    PubMed Central

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B. P.

    2016-01-01

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients. PMID:26771602

  19. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed Central

    Hagiwara, Y.; Nishio, H.; Kitoh, Y.; Takeshima, Y.; Narita, N.; Wada, H.; Yokoyama, M.; Nakamura, H.; Matsuo, M.

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. Images Figure 2 Figure 5 PMID:8279470

  20. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    SciTech Connect

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi )

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  1. HOLLYWOOD: a comparative relational database of alternative splicing.

    PubMed

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  2. MAASE: An alternative splicing database designed for supporting splicing microarray applications

    PubMed Central

    ZHENG, CHRISTINA L.; KWON, YOUNG-SOO; LI, HAI-RI; ZHANG, KUI; COUTINHO-MANSFIELD, GABRIELA; YANG, CANZHU; NAIR, T. MURLIDHARAN; GRIBSKOV, MICHAEL; FU, XIANG-DONG

    2005-01-01

    Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication. PMID:16251387

  3. Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA.

    PubMed Central

    Cheng, J; Maquat, L E

    1993-01-01

    The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to approximately 20% of normal by frameshift and nonsense mutations that cause translation to terminate at a nonsense codon within the first three-fourths of the reading frame. Results of previous studies inhibiting RNA synthesis with actinomycin D suggested that the decrease is not attributable to an increased rate of cytoplasmic mRNA decay. However, the step in TPI RNA metabolism that is altered was not defined, and the use of actinomycin D, in affecting all polymerase II-transcribed genes, could result in artifactual conclusions. In data presented here, the nonsense codon-mediated reduction in the level of TPI mRNA is shown to be characteristic of both nuclear and cytoplasmic fractions of the cell, indicating that the altered metabolic step is nucleus associated. Neither aberrancies in gene transcription nor aberrancies in RNA splicing appear to contribute to the reduction since there were no accompanying changes in the amount of nuclear run-on transcription, the level of any of the six introns in TPI pre-mRNA, or the size of processed mRNA in the nucleus. Deletion of all splice sites that reside downstream of a nonsense codon does not abrogate the reduction, indicating that the reduction takes place independently of the splicing of a downstream intron. Experiments that placed TPI gene expression under the control of the human c-fos promoter, which can be transiently activated by the addition of serum to serum-deprived cells, verified that there is no detectable effect of a nonsense codon on the turnover of cytoplasmic mRNA. Images PMID:8441420

  4. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    SciTech Connect

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-08-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

  5. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan T; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz.

  6. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.

    PubMed

    Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

    2015-01-09

    To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine.

  7. Correct splicing despite mutation of the invariant first nucleotide of a 5[prime] splice site: A possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency

    SciTech Connect

    Arredondo-Vega, F.X.; Santisteban, I.; Kelly, S.; Hershfield, M.S. ); Umetsu, D.T. ); Schlossman, C.M.

    1994-05-01

    Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. The authors have characterized the mutations responsible for ADA deficiency in siblings with disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G[sup +1][yields]A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings. 66 refs., 6 figs., 1 tab.

  8. A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism.

    PubMed Central

    Ieiri, T; Cochaux, P; Targovnik, H M; Suzuki, M; Shimoda, S; Perret, J; Vassart, G

    1991-01-01

    A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient. Images PMID:1752952

  9. Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity.

    PubMed

    Tubsuwan, Alisa; Munkongdee, Thongperm; Jearawiriyapaisarn, Natee; Boonchoy, Chanikarn; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

    2011-09-01

    Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (β-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced β(E) -globin (HBB:c.79G>A) mRNA and consequently β(+) -thalassaemia. A wide range of clinical severities in bothα- and β-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and β(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in β-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced β(E) -globin mRNA ratio in 30% of mild β-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of β(E) -globin pre-mRNA differs among β-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity.

  10. Polarization Aberrations

    NASA Technical Reports Server (NTRS)

    Mcguire, James P., Jr.; Chipman, Russell A.

    1990-01-01

    The analysis of the polarization characteristics displayed by optical systems can be divided into two categories: geometrical and physical. Geometrical analysis calculates the change in polarization of a wavefront between pupils in an optical instrument. Physical analysis propagates the polarized fields wherever the geometrical analysis is not valid, i.e., near the edges of stops, near images, in anisotropic media, etc. Polarization aberration theory provides a starting point for geometrical design and facilitates subsequent optimization. The polarization aberrations described arise from differences in the transmitted (or reflected) amplitudes and phases at interfaces. The polarization aberration matrix (PAM) is calculated for isotropic rotationally symmetric systems through fourth order and includes the interface phase, amplitude, linear diattenuation, and linear retardance aberrations. The exponential form of Jones matrices used are discussed. The PAM in Jones matrix is introduced. The exact calculation of polarization aberrations through polarization ray tracing is described. The report is divided into three sections: I. Rotationally Symmetric Optical Systems; II. Tilted and Decentered Optical Systems; and Polarization Analysis of LIDARs.

  11. Lessons from non-canonical splicing

    PubMed Central

    Ule, Jernej

    2016-01-01

    Recent improvements in experimental and computational techniques used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons, and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimises their potential to disrupt gene expression. While non-canonical splicing can lead to aberrant transcripts that cause many diseases, we also explain how it can be exploited for new therapeutic strategies. PMID:27240813

  12. PAX8 is transcribed aberrantly in cervical tumors and derived cell lines due to complex gene rearrangements.

    PubMed

    López-Urrutia, Eduardo; Pedroza-Torres, Abraham; Fernández-Retana, Jorge; De Leon, David Cantu; Morales-González, Fermín; Jacobo-Herrera, Nadia; Peralta-Zaragoza, Oscar; García-Mendez, Jorge; García-Castillo, Verónica; Bautista-Isidro, Osvaldo; Pérez-Plasencia, Carlos

    2016-07-01

    The transcription factor PAX8, a member of the paired box-containing gene family with an important role in embryogenesis of the kidney, thyroid gland and nervous system, has been described as a biomarker in tumors of the thyroid, parathyroid, kidney and thymus. The PAX8 gene gives rise to four isoforms, through alternative mRNA splicing, but the splicing pattern in tumors is not yet established. Cervical cancer has a positive expression of PAX8; however, there is no available data determining which PAX8 isoform or isoforms are present in cervical cancer tissues as well as in cervical carcinoma-derived cell lines. Instead of a differential pattern of splicing isoforms, we found numerous previously unreported PAX8 aberrant transcripts ranging from 378 to 542 bases and present in both cervical carcinoma-derived cell lines and tumor samples. This is the first report of PAX8 aberrant transcript production in cervical cancer. Reported PAX8 isoforms possess differential transactivation properties; therefore, besides being a helpful marker for detection of cancer, PAX8 isoforms can plausibly exert differential regulation properties during carcinogenesis.

  13. Abnormal Splicing of NEDD4 in Myotonic Dystrophy Type 2

    PubMed Central

    Screen, Mark; Jonson, Per Harald; Raheem, Olayinka; Palmio, Johanna; Laaksonen, Reijo; Lehtimäki, Terho; Sirito, Mario; Krahe, Ralf; Hackman, Peter; Udd, Bjarne

    2015-01-01

    Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2. PMID:24907641

  14. The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis

    PubMed Central

    Cardamone, Giulia; Paraboschi, Elvezia Maria; Rimoldi, Valeria; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

    2017-01-01

    Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB, which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease. PMID:28272342

  15. Analysis of alternative splicing events for cancer diagnosis using a multiplexing nanophotonic biosensor

    PubMed Central

    Huertas, César S.; Domínguez-Zotes, Santos; Lechuga, Laura M.

    2017-01-01

    Personalized medicine is a promising tool not only for prevention, screening and development of more efficient treatment strategies, but also for diminishing the side effects caused by current therapies. Deciphering gene regulation pathways provides a reliable prognostic analysis to elucidate the origin of grave diseases and facilitate the selection of the most adequate treatment for each individual. Alternative splicing of mRNA precursors is one of these gene regulation pathways and enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression constituting a relevant and innovative class of biomarker. Herein we report a highly selective and sensitive nanophotonic biosensor based on the direct monitoring of the aberrant alternative splicing of Fas gene. Unlike conventional methods, the nanobiosensor performs a real-time detection of the specific isoforms in the fM-pM range without any cDNA synthesis or PCR amplification requirements. The nanobiosensor has been proven isoform-specific with no crosshybridization, greatly minimizing detection biases. The demonstrated high sensitivity and specificity make our nanobiosensor ideal for examining significant tumor-associated expression shifts of alternatively spliced isoforms for the early and accurate theranostics of cancer. PMID:28120920

  16. Understanding splicing regulation through RNA splicing maps.

    PubMed

    Witten, Joshua T; Ule, Jernej

    2011-03-01

    Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

  17. Pathophysiology of MDS: genomic aberrations.

    PubMed

    Ichikawa, Motoshi

    Myelodysplastic syndromes (MDS) are characterized by clonal proliferation of hematopoietic stem/progenitor cells and their apoptosis, and show a propensity to progress to acute myelogenous leukemia (AML). Although MDS are recognized as neoplastic diseases caused by genomic aberrations of hematopoietic cells, the details of the genetic abnormalities underlying disease development have not as yet been fully elucidated due to difficulties in analyzing chromosomal abnormalities. Recent advances in comprehensive analyses of disease genomes including whole-genome sequencing technologies have revealed the genomic abnormalities in MDS. Surprisingly, gene mutations were found in approximately 80-90% of cases with MDS, and the novel mutations discovered with these technologies included previously unknown, MDS-specific, mutations such as those of the genes in the RNA-splicing machinery. It is anticipated that these recent studies will shed new light on the pathophysiology of MDS due to genomic aberrations.

  18. Can the HIV-1 splicing machinery be targeted for drug discovery?

    PubMed

    Dlamini, Zodwa; Hull, Rodney

    2017-01-01

    HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed.

  19. Can the HIV-1 splicing machinery be targeted for drug discovery?

    PubMed Central

    Dlamini, Zodwa; Hull, Rodney

    2017-01-01

    HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. PMID:28331370

  20. mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

    NASA Astrophysics Data System (ADS)

    Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

    2016-11-01

    Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

  1. mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

    PubMed Central

    Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

    2016-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction. PMID:27869204

  2. mRNA changes in nucleus accumbens related to methamphetamine addiction in mice.

    PubMed

    Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L K; Chen, Teng

    2016-11-21

    Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

  3. Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy.

    PubMed

    Martorell, L; Corrales, I; Ramirez, L; Parra, R; Raya, A; Barquinero, J; Vidal, F

    2015-03-01

    Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

  4. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  5. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

    PubMed Central

    Bergsma, Atze J; in ‘t Groen, Stijn LM; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, WWM Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  6. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  7. [Alternative splicing regulation: implications in cancer diagnosis and treatment].

    PubMed

    Martínez-Montiel, Nancy; Rosas-Murrieta, Nora; Martínez-Contreras, Rebeca

    2015-04-08

    The accurate expression of the genetic information is regulated by processes like mRNA splicing, proposed after the discoveries of Phil Sharp and Richard Roberts, who demonstrated the existence of intronic sequences, present in almost every structural eukaryotic gene, which should be precisely removed. This intron removal is called "splicing", which generates different proteins from a single mRNA, with different or even antagonistic functions. We currently know that alternative splicing is the most important source of protein diversity, given that 70% of the human genes undergo splicing and that mutations causing defects in this process could originate up to 50% of genetic diseases, including cancer. When these defects occur in genes involved in cell adhesion, proliferation and cell cycle regulation, there is an impact on cancer progression, rising the opportunity to diagnose and treat some types of cancer according to a particular splicing profile.

  8. Functional studies on the ATM intronic splicing processing element.

    PubMed

    Lewandowska, Marzena A; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

  9. Functional studies on the ATM intronic splicing processing element

    PubMed Central

    Lewandowska, Marzena A.; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E.; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing. PMID:16030351

  10. Evolutionary Insights into RNA trans-Splicing in Vertebrates

    PubMed Central

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-01-01

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

  11. Evolutionary Insights into RNA trans-Splicing in Vertebrates.

    PubMed

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-03-10

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint.

  12. Tau mis-splicing in the pathogenesis of neurodegenerative disorders

    PubMed Central

    Park, Sun Ah; Ahn, Sang Il; Gallo, Jean-Marc

    2016-01-01

    Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413] PMID:27222125

  13. A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome.

    PubMed

    Inturi, Raviteja; Wäneskog, Marcus; Vlachakis, Dimitrios; Ali, Yeasmeen; Ek, Pia; Punga, Tanel; Bjerling, Pernilla

    2014-12-01

    Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.

  14. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  15. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    PubMed

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  16. Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

    PubMed

    Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

    2004-03-17

    Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

  17. The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing

    PubMed Central

    Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

    2016-01-01

    ABSTRACT Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

  18. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  19. Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics

    PubMed Central

    Barbon, Elena; Ferrarese, Mattia; van Wittenberghe, Laetitia; Sanatine, Peggy; Ronzitti, Giuseppe; Collaud, Fanny; Colella, Pasqualina; Pinotti, Mirko; Mingozzi, Federico

    2016-01-01

    Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies. PMID:27898092

  20. Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

    PubMed

    Carothers, A M; Urlaub, G; Grunberger, D; Chasin, L A

    1993-08-01

    Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a

  1. Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

    PubMed

    Aznarez, Isabel; Chan, Elayne M; Zielenski, Julian; Blencowe, Benjamin J; Tsui, Lap-Chee

    2003-08-15

    Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

  2. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout

    PubMed Central

    Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level. PMID:27983621

  3. Targeting RNA-splicing for SMA treatment.

    PubMed

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

    The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease.

  4. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

    PubMed

    Darman, Rachel B; Seiler, Michael; Agrawal, Anant A; Lim, Kian H; Peng, Shouyong; Aird, Daniel; Bailey, Suzanna L; Bhavsar, Erica B; Chan, Betty; Colla, Simona; Corson, Laura; Feala, Jacob; Fekkes, Peter; Ichikawa, Kana; Keaney, Gregg F; Lee, Linda; Kumar, Pavan; Kunii, Kaiko; MacKenzie, Crystal; Matijevic, Mark; Mizui, Yoshiharu; Myint, Khin; Park, Eun Sun; Puyang, Xiaoling; Selvaraj, Anand; Thomas, Michael P; Tsai, Jennifer; Wang, John Y; Warmuth, Markus; Yang, Hui; Zhu, Ping; Garcia-Manero, Guillermo; Furman, Richard R; Yu, Lihua; Smith, Peter G; Buonamici, Silvia

    2015-11-03

    Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

  5. Skew aberration: a form of polarization aberration.

    PubMed

    Yun, Garam; Crabtree, Karlton; Chipman, Russell A

    2011-10-15

    We define a new class of aberration, skew aberration, which is a component of polarization aberration. Skew aberration is an intrinsic rotation of polarization states due to the geometric transformation of local coordinates, independent of coatings and interface polarization. Skew aberration in a radially symmetric system has the form of a circular retardance tilt plus coma aberration. Skew aberration causes undesired polarization distribution in the exit pupil. We demonstrate statistics on skew aberration of 2383 optical systems described in Code V's U.S. patent library [Code V Version 10.3 (Synopsys, 2011), pp. 22-24]; the mean skew aberration is 0.89° and the standard deviation is 1.37°. The maximum skew aberration found is 17.45° and the minimum is -11.33°. U.S. patent 2,896,506, which has ±7.01° of skew aberration, is analyzed in detail. Skew aberration should be of concern in microlithography optics and other high NA and large field of view optical systems.

  6. Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver*

    PubMed Central

    Walsh, Callee M.; Suchanek, Amanda L.; Cyphert, Travis J.; Kohan, Alison B.; Szeszel-Fedorowicz, Wioletta; Salati, Lisa M.

    2013-01-01

    Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing. PMID:23233666

  7. Serine arginine splicing factor 3 is involved in enhanced splicing of glucose-6-phosphate dehydrogenase RNA in response to nutrients and hormones in liver.

    PubMed

    Walsh, Callee M; Suchanek, Amanda L; Cyphert, Travis J; Kohan, Alison B; Szeszel-Fedorowicz, Wioletta; Salati, Lisa M

    2013-01-25

    Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.

  8. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy. 

  9. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-05

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease.

  10. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  11. Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2.

    PubMed Central

    Deutscher, S L; Bhat, B M; Pursley, M H; Cladaras, C; Wold, W S

    1985-01-01

    Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site. Images PMID:2412208

  12. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  13. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  14. Quantification of pre-mRNA escape rate and synergy in splicing

    PubMed Central

    Bonde, Marie Mi; Voegeli, Sylvia; Baudrimont, Antoine; Séraphin, Bertrand; Becskei, Attila

    2014-01-01

    Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks. PMID:25352554

  15. Mechanisms and Regulation of Alternative Pre-mRNA Splicing

    PubMed Central

    Lee, Yeon

    2015-01-01

    Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

  16. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

    PubMed

    Listerman, Imke; Sapra, Aparna K; Neugebauer, Karla M

    2006-09-01

    Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

  17. The Nuclear Ribonucleoprotein SmD1 Interplays with Splicing, RNA Quality Control, and Posttranscriptional Gene Silencing in Arabidopsis.

    PubMed

    Elvira-Matelot, Emilie; Bardou, Florian; Ariel, Federico; Jauvion, Vincent; Bouteiller, Nathalie; Le Masson, Ivan; Cao, Jun; Crespi, Martin D; Vaucheret, Hervé

    2016-02-01

    RNA quality control (RQC) eliminates aberrant RNAs based on their atypical structure, whereas posttranscriptional gene silencing (PTGS) eliminates both aberrant and functional RNAs through the sequence-specific action of short interfering RNAs (siRNAs). The Arabidopsis thaliana mutant smd1b was identified in a genetic screen for PTGS deficiency, revealing the involvement of SmD1, a component of the Smith (Sm) complex, in PTGS. The smd1a and smd1b single mutants are viable, but the smd1a smd1b double mutant is embryo-lethal, indicating that SmD1 function is essential. SmD1b resides in nucleoli and nucleoplasmic speckles, colocalizing with the splicing-related factor SR34. Consistent with this, the smd1b mutant exhibits intron retention at certain endogenous mRNAs. SmD1 binds to RNAs transcribed from silenced transgenes but not nonsilenced ones, indicating a direct role in PTGS. Yet, mutations in the RQC factors UPFRAMESHIFT3, EXORIBONUCLEASE2 (XRN2), XRN3, and XRN4 restore PTGS in smd1b, indicating that SmD1 is not essential for but rather facilitates PTGS. Moreover, the smd1b mtr4 double mutant is embryo-lethal, suggesting that SmD1 is essential for mRNA TRANSPORT REGULATOR4-dependent RQC. These results indicate that SmD1 interplays with splicing, RQC, and PTGS. We propose that SmD1 facilitates PTGS by protecting transgene-derived aberrant RNAs from degradation by RQC in the nucleus, allowing sufficient amounts to enter cytoplasmic siRNA bodies to activate PTGS.

  18. Characterisation of unclassified variants in the BRCA1/2 genes with a putative effect on splicing.

    PubMed

    Brandão, Rita Dias; van Roozendaal, Kees; Tserpelis, Demis; Gómez García, Encarna; Blok, Marinus J

    2011-10-01

    A subset of the unclassified variants (UVs) identified during genetic screening of BRCA1/2 genes may affect splicing. We assessed at RNA level the effect of four BRCA1 and ten BRCA2 UVs with a putative splice effect, as predicted in silico. The variants selected for this study were beyond the positions -1, -2 or +1, +2 from the exon, and were not previously described (n = 8) or their effect on splicing was not assessed previously (n = 6). Lymphocytes from UV carriers and healthy controls were cultured and treated with puromycin to prevent nonsense-mediated mRNA decay. The relative contribution of each allele to the various transcripts was assessed using combinations of allele-specific and transcript-specific primers. BRCA2 c.425G>T, c.7976+3_7976+4del and c.8754+3G>C give rise to aberrant transcripts BRCA2Δ4, BRCA2Δ17 and retention of 46nt of intron 21, respectively, and were considered pathogenic. BRCA1 c.4987-3C>G gives rise to BRCA1Δ17 that is likely pathogenic; however, residual expression of the full-length transcript from the variant allele could not be excluded. BRCA1 c.692C>T, c.693G>A and BRCA2 c.6935A>T, besides expressing the full-length transcript, increased expression of BRCA1Δ11 and BRCA2Δ12, respectively. As these are naturally occurring isoforms, also observed in controls, the clinical relevance is unclear. The seven remaining UVs did not affect splicing and three intronic variants were therefore classified as neutral. In conclusion, the RNA analysis results clarified the clinical relevance of 6 of the 14 studied UVs and thereby greatly improve the genetic counselling of high-risk breast/ovarian cancer patients carrying these classified variants.

  19. Influenza Virus mRNA Trafficking Through Host Nuclear Speckles

    PubMed Central

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W.; García-Sastre, Adolfo; Fontoura, Beatriz M.A.

    2016-01-01

    Influenza A virus is a human pathogen whose genome is comprised of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. Since nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression. PMID:27347430

  20. Splicing plastic optical fibers

    NASA Astrophysics Data System (ADS)

    Carson, Susan D.; Salazar, Roberto A.

    1991-12-01

    Polymethylmethacrylate (PMMA) plastic optical fiber (500 micrometers diameter, fluoropolymer cladding) has been spliced using a fused silica sleeve and a variety of solvent/PMMA solutions as adhesives. Mechanical splicing using index matching fluid has also been investigated. To ensure good bonding and minimize scattering, fiber ends are polished prior to application of adhesive. Using an LED ((lambda) max approximately 640 nm), losses are routinely less than 1.0 dB/splice, and some adhesive formulations have exhibited losses as low as 0.2 dB/splice. Five-meter fibers with as many as ten splices/fiber have been monitored over a period of several months. No fiber has exhibited an increase in optical loss with time.

  1. RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

    PubMed Central

    Doktor, Thomas Koed; Hua, Yimin; Andersen, Henriette Skovgaard; Brøner, Sabrina; Liu, Ying Hsiu; Wieckowska, Anna; Dembic, Maja; Bruun, Gitte Hoffmann; Krainer, Adrian R.; Andresen, Brage Storstein

    2017-01-01

    Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by insufficient levels of the Survival of Motor Neuron (SMN) protein. SMN is expressed ubiquitously and functions in RNA processing pathways that include trafficking of mRNA and assembly of snRNP complexes. Importantly, SMA severity is correlated with decreased snRNP assembly activity. In particular, the minor spliceosomal snRNPs are affected, and some U12-dependent introns have been reported to be aberrantly spliced in patient cells and animal models. SMA is characterized by loss of motor neurons, but the underlying mechanism is largely unknown. It is likely that aberrant splicing of genes expressed in motor neurons is involved in SMA pathogenesis, but increasing evidence indicates that pathologies also exist in other tissues. We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5. PMID:27557711

  2. Identification of recurrent regulated alternative splicing events across human solid tumors

    PubMed Central

    Danan-Gotthold, Miri; Golan-Gerstl, Regina; Eisenberg, Eli; Meir, Keren; Karni, Rotem; Levanon, Erez Y.

    2015-01-01

    Cancer is a complex disease that involves aberrant gene expression regulation. Discriminating the modified expression patterns driving tumor biology from the many that have no or little contribution is important for understanding cancer molecular basis. Recurrent deregulation patterns observed in multiple cancer types are enriched for such driver events. Here, we studied splicing alterations in hundreds of matched tumor and normal RNA-seq samples of eight solid cancer types. We found hundreds of cassette exons for which splicing was altered in multiple cancer types and identified a set of highly frequent altered splicing events. Specific splicing regulators, including RBFOX2, MBNL1/2 and QKI, appear to account for many splicing alteration events in multiple cancer types. Together, our results provide a first global analysis of regulated splicing alterations in cancer and identify common events with a potential causative role in solid tumor development. PMID:25908786

  3. Splicing factor gene mutations in the myelodysplastic syndromes: impact on disease phenotype and therapeutic applications.

    PubMed

    Pellagatti, Andrea; Boultwood, Jacqueline

    2017-01-01

    Splicing factor gene mutations are the most frequent mutations found in patients with the myeloid malignancy myelodysplastic syndrome (MDS), suggesting that spliceosomal dysfunction plays a major role in disease pathogenesis. The aberrantly spliced target genes and deregulated cellular pathways associated with the commonly mutated splicing factor genes in MDS (SF3B1, SRSF2 and U2AF1) are being identified, illuminating the molecular mechanisms underlying MDS. Emerging data from mouse modeling studies indicate that the presence of splicing factor gene mutations can lead to bone marrow hematopoietic stem/myeloid progenitor cell expansion, impaired hematopoiesis and dysplastic differentiation that are hallmarks of MDS. Importantly, recent evidence suggests that spliceosome inhibitors and splicing modulators may have therapeutic value in the treatment of splicing factor mutant myeloid malignancies.

  4. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

    PubMed

    Selvanathan, Saravana P; Graham, Garrett T; Erkizan, Hayriye V; Dirksen, Uta; Natarajan, Thanemozhi G; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T; Ljungman, Mats E; Wu, Cathy H; Lawlor, Elizabeth R; Üren, Aykut; Toretsky, Jeffrey A

    2015-03-17

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

  5. Chicago aberration correction work.

    PubMed

    Beck, V D

    2012-12-01

    The author describes from his personal involvement the many improvements to electron microscopy Albert Crewe and his group brought by minimizing the effects of aberrations. The Butler gun was developed to minimize aperture aberrations in a field emission electron gun. In the 1960s, Crewe anticipated using a spherical aberration corrector based on Scherzer's design. Since the tolerances could not be met mechanically, a method of moving the center of the octopoles electrically was developed by adding lower order multipole fields. Because the corrector was located about 15 cm ahead of the objective lens, combination aberrations would arise with the objective lens. This fifth order aberration would then limit the aperture of the microscope. The transformation of the off axis aberration coefficients of a round lens was developed and a means to cancel anisotropic coma was developed. A new method of generating negative spherical aberration was invented using the combination aberrations of hexapoles. Extensions of this technique to higher order aberrations were developed. An electrostatic electron mirror was invented, which allows the cancellation of primary spherical aberration and first order chromatic aberration. A reduction of chromatic aberration by two orders of magnitude was demonstrated using such a system.

  6. An XPA gene splicing mutation resulting in trace protein expression in an elderly xeroderma pigmentosum group A patient without neurological abnormalities.

    PubMed

    Takahashi, Y; Endo, Y; Kusaka, A; Nakamaura, S; Nakazawa, Y; Ogi, T; Uryu, M; Tsuji, M; Furue, M; Moriwaki, S

    2016-09-07

    A certain relationship between XPA gene mutations and the severity of symptoms has been observed in patients with xeroderma pigmentosum group A (XP-A). Patients with mutations within the DNA-binding domain usually exhibit severe symptoms, whereas splicing mutations in the same domain sometimes cause very mild symptoms. This inconsistency can be explained by a small amount of functional XPA protein produced from normally spliced transcripts. We herein report the case of an adult Japanese XP-A patient with unusually mild symptoms. We identified a homozygous c.529G>A mutation in exon 4 of the XPA gene, which resulted in aberrant splicing with a 29-bp deletion in exon 4 causing a frameshift. Intact mRNA was observable, but a Western blot analysis failed to detect any normal XPA protein. We therefore evaluated the DNA repair capacity in normal cells in which the XPA expression was artificially diminished. The repair capacity was still present in cells with trace levels of the XPA protein. The repair capacity of the cells derived from our patient with mild symptoms was poor by comparison, but still significant compared to that of the cells derived from an XP-A patient with severe symptoms. These results provide strong evidence that a trace level of XPA protein can still exert a relatively strong repair capacity, resulting in only a mild phenotype. This article is protected by copyright. All rights reserved.

  7. Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1)

    PubMed Central

    Chung, Felicia Fei-Lei; Tan, Perry Faith Tze Ming; Raja, Vijay Joseph; Tan, Boon-Shing; Lim, Kuan-Hon; Kam, Toh-Seok; Hii, Ling-Wei; Tan, Si Hoey; See, Sze-Jia; Tan, Yuen-Fen; Wong, Li-Zhe; Yam, Wai Keat; Mai, Chun Wai; Bradshaw, Tracey D.; Leong, Chee-Onn

    2017-01-01

    Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization. PMID:28198434

  8. Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation

    PubMed Central

    2013-01-01

    Background Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups. Case presentation In this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475 + 1G > C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa. Conclusion This report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling. PMID:23506628

  9. SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis.

    PubMed

    Feng, Jinlin; Li, Jingjing; Gao, Zhaoxu; Lu, Yaru; Yu, Junya; Zheng, Qian; Yan, Shuning; Zhang, Wenjiao; He, Hang; Ma, Ligeng; Zhu, Zhengge

    2015-07-01

    Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

  10. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  11. Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).

    PubMed

    Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

    2013-05-01

    Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

  12. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

    NASA Astrophysics Data System (ADS)

    Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  13. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing.

    PubMed

    Nguyen, Anh H; Lee, Jong Uk; Sim, Sang Jun

    2016-02-28

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ∼29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  14. The Role of Canonical and Noncanonical Pre-mRNA Splicing in Plant Stress Responses

    PubMed Central

    Dubrovina, A. S.; Kiselev, K. V.; Zhuravlev, Yu. N.

    2013-01-01

    Plants are sessile organisms capable of adapting to various environmental constraints, such as high or low temperatures, drought, soil salinity, or pathogen attack. To survive the unfavorable conditions, plants actively employ pre-mRNA splicing as a mechanism to regulate expression of stress-responsive genes and reprogram intracellular regulatory networks. There is a growing evidence that various stresses strongly affect the frequency and diversity of alternative splicing events in the stress-responsive genes and lead to an increased accumulation of mRNAs containing premature stop codons, which in turn have an impact on plant stress response. A number of studies revealed that some mRNAs involved in plant stress response are spliced counter to the traditional conception of alternative splicing. Such noncanonical mRNA splicing events include trans-splicing, intraexonic deletions, or variations affecting multiple exons and often require short direct repeats to occur. The noncanonical alternative splicing, along with common splicing events, targets the spliced transcripts to degradation through nonsense-mediated mRNA decay or leads to translation of truncated proteins. Investigation of the diversity, biological consequences, and mechanisms of the canonical and noncanonical alternative splicing events will help one to identify those transcripts which are promising for using in genetic engineering and selection of stress-tolerant plants. PMID:23509698

  15. Hydrogen peroxide triggers a novel alternative splicing of arsenic (+3 oxidation state) methyltransferase gene.

    PubMed

    Sumi, Daigo; Takeda, Chieri; Yasuoka, Daiki; Himeno, Seiichiro

    2016-11-04

    We previously reported that two splicing variants of human AS3MT mRNA, exon-3 skipping form (Δ3) and exons-4 and -5 skipping form (Δ4,5), were detected in HepG2 cells and that both variants lacked arsenic methylation activity (Sumi et al., 2011). Here we studied whether hydrogen peroxide (H2O2) triggers alternative splicing of AS3MT mRNA. The results showed that exposure of HepG2 cells to H2O2 resulted in increased levels of a novel spliced form skipping exon-3 to exon-10 (Δ3-10) in an H2O2-concentration-dependent manner, although no change was detected in the mRNA levels of Δ3 AS3MT. We found decreased protein levels of serine/arginine-rich 40 (SRp40), which we determined to be a candidate splice factor for controlling the splicing of AS3MT mRNA. We next compared the amounts of methylated arsenic metabolites between control and H2O2-exposed HepG2 cells after the addition of arsenite as a substance. The results showed lower levels of methylated arsenic metabolites in HepG2 cells exposed to H2O2. These data suggest that the splicing of AS3MT pre-mRNA was disconcerted by oxidative stress and that abnormal alternative splicing of AS3MT mRNA may affect arsenic methylation ability.

  16. Regulation of chemoresistance via alternative messenger RNA splicing.

    PubMed

    Eblen, Scott T

    2012-04-15

    The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.

  17. RNA helicases in splicing.

    PubMed

    Cordin, Olivier; Beggs, Jean D

    2013-01-01

    In eukaryotic cells, introns are spliced from pre-mRNAs by the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. There is evidence that the various helicases are functionally and physically connected with each other and with many other factors in the spliceosome. Understanding the dynamics of those interactions is essential to comprehend the mechanism and regulation of normal as well as of pathological splicing. This review focuses on recent advances in the characterization of the splicing helicases and their interactions, and highlights the deep integration of splicing helicases in global mRNP biogenesis pathways.

  18. The Role of Alternative Splicing in Breast Cancer Progression

    DTIC Science & Technology

    2007-09-01

    tumorigenesis Reportable Outcomes: -portions of the work have been chosen for an oral presentation at this year’s Cold Spring Harbor “Eukaryotic mRNA...splicing alterations. References: N/A Appendices: Copy of abstract of the work presented during the Cold Spring Harbor “Eukaryotic mRNA Processing” meeting, August 22-26, 2007

  19. Alternative splicing and genomic structure of the Wilms tumor gene WT1

    SciTech Connect

    Haber, D.A. Massachusetts General Hospital Cancer Center, Charlestown ); Sohn, R.L.; Buckler, A.J.; Pelletier, J.; Call, K.M.; Housman, D.E. )

    1991-11-01

    The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5{prime} splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggest that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.

  20. Interpreting Chromosome Aberration Spectra

    NASA Technical Reports Server (NTRS)

    Levy, Dan; Reeder, Christopher; Loucas, Bradford; Hlatky, Lynn; Chen, Allen; Cornforth, Michael; Sachs, Rainer

    2007-01-01

    Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH ( multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.

  1. Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

    Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.

  2. Splicing defects caused by exonic mutations in PKD1 as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease.

    PubMed

    Claverie-Martin, Felix; Gonzalez-Paredes, Francisco J; Ramos-Trujillo, Elena

    2015-01-01

    The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

  3. Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast.

    PubMed Central

    Sawa, H; Abelson, J

    1992-01-01

    U6 small nuclear RNA (snRNA) is an essential factor in mRNA splicing. On the basis of the high conservation of its sequence, it has been proposed that U6 snRNA may function catalytically during the splicing reaction. If this is the case, it is likely that U6 snRNA interacts with the splice sites in the spliceosome to catalyze the reaction. We have used UV crosslinking to analyze the interactions of U6 snRNA with the splicing substrates during the yeast splicing reaction. Crosslinked products in which the central region of U6 snRNA was joined to the 5' splice site region of mRNA precursor and lariat intermediate were identified. The crosslinking sites were precisely located in one of these products. The results suggest a possible base-pairing interaction between U6 snRNA and the 5' splice site of the mRNA precursor. Images PMID:1333604

  4. Identification and characterization of NF1 splicing mutations in Korean patients with neurofibromatosis type 1.

    PubMed

    Jang, Mi-Ae; Kim, Young-Eun; Kim, Sun Kyung; Lee, Myoung-Keun; Kim, Jong-Won; Ki, Chang-Seok

    2016-08-01

    Neurofibromatosis type I (NF1) is an autosomal dominant genetic disorder caused by NF1 mutations. Although mutations affecting mRNA splicing are the most common molecular defects in NF1, few studies have analyzed genomic DNA (gDNA)-mRNA correlations in Korean NF1 patients. In this study, we investigated 28 unrelated NF1 patients who showed splicing alterations in reverse transcription-PCR of NF1 mRNA and identified 24 different NF1 splicing mutations, 9 of which were novel. These mutations can be categorized into five groups: exon skipping resulting from mutations at authentic 5' and 3' splice sites (type I, 46%), cryptic exon inclusion caused by deep intronic mutations (type II, 8%), creation of new splice sites causing loss of exonic sequences (type III, 8%), activation of cryptic splice sites due to disruption of authentic splice sites (type IV, 25%) and exonic sequence alterations causing exon skipping (type V, 13%). In total, 42% of all splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites, making it difficult to identify the correct mutation sites at the gDNA level. These results add to the mutational spectrum of NF1 and further elucidate the gDNA-mRNA correlations of NF1 mutations.

  5. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

    SciTech Connect

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

    2014-04-25

    Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

  6. Development and media regulate alternative splicing of a methyltransferase pre-mRNA in Monascus pilosus.

    PubMed

    Zhang, Ming-Yong; Miyake, Tsuyoshi

    2009-05-27

    Two alternatively spliced mRNAs (d- and l-MpLaeA) of a methyltransferase gene (MpLaeA) were identified from Monascus pilosus IFO4520 and its mutant MK-1. Alternative splicing of the MpLaeA pre-mRNA occurred in the 5'-untranslated region (5'-UTR). The alternative splicing patterns of MpLaeA were regulated by the fungal growth stage and the principal nutrients: that is, the short l-MpLaeA mRNA was a constitutive transcript at all growth stages and different carbon or nitrogen sources, but the glutamate and NaNO(3) as main nitrogen source could up-regulate the long d-MpLaeA mRNA form. The long spliced 5'-UTR of d-MpLaeA blocked GFP expression in Escherichia coli , suggesting that d-MpLaeA mRNA was an ineffective spliced mRNA. Down-regulation of MpLaeA by transgenic antisense d-MpLaeA cDNA resulted in decreasing synthesis of monacolin K in M. pilosus. This suggested that the alternative splicing of MpLaeA mRNA might regulate the synthesis of monacolin K.

  7. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

  8. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

    PubMed Central

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-01-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations. PMID:21407258

  9. SpliceDisease database: linking RNA splicing and disease.

    PubMed

    Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua

    2012-01-01

    RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation, we give direct links of the entry to the respective position/region in the genome browser. The users can freely browse, search and download the data in SpliceDisease at http://cmbi.bjmu.edu.cn/sdisease.

  10. Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin.

    PubMed

    Mano, S; Hayashi, M; Nishimura, M

    1999-02-01

    Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.

  11. Workflow for Genome-Wide Determination of Pre-mRNA Splicing Efficiency from Yeast RNA-seq Data

    PubMed Central

    Folk, Petr

    2016-01-01

    Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5′ and 3′ splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions. PMID:28050562

  12. Workflow for Genome-Wide Determination of Pre-mRNA Splicing Efficiency from Yeast RNA-seq Data.

    PubMed

    Převorovský, Martin; Hálová, Martina; Abrhámová, Kateřina; Libus, Jiří; Folk, Petr

    2016-01-01

    Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

  13. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  14. The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

    NASA Astrophysics Data System (ADS)

    Choudhury, Rajarshi; Roy, Sreerupa Ghose; Tsai, Yihsuan S.; Tripathy, Ashutosh; Graves, Lee M.; Wang, Zefeng

    2014-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.

  15. The leader region of Laminin B1 mRNA confers cap-independent translation.

    PubMed

    Petz, Michaela; Kozina, Daniela; Huber, Heidemarie; Siwiec, Tanja; Seipelt, Joachim; Sommergruber, Wolfgang; Mikulits, Wolfgang

    2007-01-01

    Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5'-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5'-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes.

  16. Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

    PubMed

    Teresa-Rodrigo, María E; Eckhold, Juliane; Puisac, Beatriz; Dalski, Andreas; Gil-Rodríguez, María C; Braunholz, Diana; Baquero, Carolina; Hernández-Marcos, María; de Karam, Juan C; Ciero, Milagros; Santos-Simarro, Fernando; Lapunzina, Pablo; Wierzba, Jolanta; Casale, César H; Ramos, Feliciano J; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J; Pié, Juan

    2014-06-10

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

  17. Functional Characterization of NIPBL Physiological Splice Variants and Eight Splicing Mutations in Patients with Cornelia de Lange Syndrome

    PubMed Central

    Teresa-Rodrigo, María E.; Eckhold, Juliane; Puisac, Beatriz; Dalski, Andreas; Gil-Rodríguez, María C.; Braunholz, Diana; Baquero, Carolina; Hernández-Marcos, María; de Karam, Juan C.; Ciero, Milagros; Santos-Simarro, Fernando; Lapunzina, Pablo; Wierzba, Jolanta; Casale, César H.; Ramos, Feliciano J.; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J.; Pié, Juan

    2014-01-01

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame. PMID:24918291

  18. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes

    PubMed Central

    Machinaga, Akihito; Ishihara, Syuhei; Shirai, Akiko; Takase-Yoden, Sayaka

    2016-01-01

    Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5′ long terminal repeat (LTR), 5′ leader sequence, gag, pol, env, and 3′ LTR. Transcription from proviral DNA begins from the R region of the 5′ LTR and ends at the polyadenylation signal located at the R region of the other end of the 3′ LTR. There is a 5′ splice site in the 5′ leader sequence and a 3′ splice site at the 3′ end of the pol region. Both full-length unspliced mRNAs and a singly spliced mRNA (env-mRNA) are produced in MLV-infected cells. The MLV Env protein plays important roles both in viral adsorption to host cells and in neuropathogenic disease in MLV-infected mice and rats. Understanding the regulatory mechanisms controlling Env expression is important for determining the functions of the Env protein. We have previously shown that splicing increases env-mRNA stability and translation efficiency. Generally, mRNA polysome formation correlates with translation efficiency. Therefore, here we investigated the effects of env-mRNA splicing on polysome formation to identify mechanisms for Env up-regulation due to splicing. We performed polysome profile analyses using Env-expression plasmids producing spliced or unspliced env-mRNA and showed that the former formed polysomes more efficiently than the latter. Thus, splicing of env-mRNA facilitated polysome formation, suggesting that this contributes to up-regulation of Env expression. We replaced the env region of the expression plasmids with a luciferase (luc) gene, and found that in this case both unspliced and spliced luc-mRNA formed polysomes to a similar extent. Thus, we conclude that whether mRNA polysome formation is affected by splicing depends on the structure of gene in question. PMID:26909075

  19. High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

    PubMed

    Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

    2010-05-01

    Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

  20. The Involvement of Splicing Factor hnRNP A1 in UVB-Induced Alternative Splicing of hdm2.

    PubMed

    Feng, Jianguo; Li, Li; Tong, Lingying; Tang, Liling; Wu, Shiyong

    2016-01-12

    Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 via increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA. This article is protected by copyright. All rights reserved.

  1. Modulation of alternative splicing with chemical compounds in new therapeutics for human diseases.

    PubMed

    Ohe, Kenji; Hagiwara, Masatoshi

    2015-04-17

    Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the "genome code," have been found to result in an aberrant mis-spliced "transcript code" with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced "transcript code". We will briefly mention about how to obtain splicing-modifying-compounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acid-based strategies. The information on their involvement in mis-splicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy.

  2. MET receptor juxtamembrane exon 14 alternative spliced variant: novel cancer genomic predictive biomarker.

    PubMed

    Ma, Patrick C

    2015-08-01

    Clinical studies on MET-targeting cancer therapeutics have yielded mixed results in recent years, and MET-relevant predictive biomarkers remain elusive. New studies now reveal METex14 alternative splicing aberrations to represent potential predictive cancer genomic biomarker, hence renewing optimism and directions in the quest for optimized MET-targeting personalized cancer therapy.

  3. Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment.

    PubMed

    Ahmed, Iltaf; Buchert, Rebecca; Zhou, Mi; Jiao, Xinfu; Mittal, Kirti; Sheikh, Taimoor I; Scheller, Ute; Vasli, Nasim; Rafiq, Muhammad Arshad; Brohi, M Qasim; Mikhailov, Anna; Ayaz, Muhammad; Bhatti, Attya; Sticht, Heinrich; Nasr, Tanveer; Carter, Melissa T; Uebe, Steffen; Reis, André; Ayub, Muhammad; John, Peter; Kiledjian, Megerditch; Vincent, John B; Jamra, Rami Abou

    2015-06-01

    There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

  4. Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment

    PubMed Central

    Ahmed, Iltaf; Buchert, Rebecca; Zhou, Mi; Jiao, Xinfu; Mittal, Kirti; Sheikh, Taimoor I.; Scheller, Ute; Vasli, Nasim; Rafiq, Muhammad Arshad; Brohi, M. Qasim; Mikhailov, Anna; Ayaz, Muhammad; Bhatti, Attya; Sticht, Heinrich; Nasr, Tanveer; Carter, Melissa T.; Uebe, Steffen; Reis, André; Ayub, Muhammad; John, Peter; Kiledjian, Megerditch; Vincent, John B.; Jamra, Rami Abou

    2015-01-01

    There are two known mRNA degradation pathways, 3′ to 5′ and 5′ to 3′. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3′ to 5′ exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5′ to 3′ degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects. PMID:25701870

  5. CUG-BP1 regulates RyR1 ASI alternative splicing in skeletal muscle atrophy.

    PubMed

    Tang, Yinglong; Wang, Huiwen; Wei, Bin; Guo, Yuting; Gu, Lei; Yang, Zhiguang; Zhang, Qing; Wu, Yanyun; Yuan, Qi; Zhao, Gang; Ji, Guangju

    2015-11-04

    RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease.

  6. Transcription and splicing: when the twain meet.

    PubMed

    Brody, Yehuda; Shav-Tal, Yaron

    2011-01-01

    Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

  7. Trans-splicing improvement by the combined application of antisense strategies.

    PubMed

    Koller, Ulrich; Hainzl, Stefan; Kocher, Thomas; Hüttner, Clemens; Klausegger, Alfred; Gruber, Christina; Mayr, Elisabeth; Wally, Verena; Bauer, Johann W; Murauer, Eva M

    2015-01-06

    Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  8. A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1

    PubMed Central

    Hollander, Dror; Donyo, Maya; Atias, Nir; Mekahel, Keren; Melamed, Zeev; Yannai, Sivan; Lev-Maor, Galit; Shilo, Asaf; Schwartz, Schraga; Barshack, Iris; Sharan, Roded; Ast, Gil

    2016-01-01

    Splicing aberrations are prominent drivers of cancer, yet the regulatory pathways controlling them are mostly unknown. Here we develop a method that integrates physical interaction, gene expression, and alternative splicing data to construct the largest map of transcriptomic and proteomic interactions leading to cancerous splicing aberrations defined to date, and identify driver pathways therein. We apply our method to colon adenocarcinoma and non-small-cell lung carcinoma. By focusing on colon cancer, we reveal a novel tumor-favoring regulatory pathway involving the induction of the transcription factor MYC by the transcription factor ELK1, as well as the subsequent induction of the alternative splicing factor PTBP1 by both. We show that PTBP1 promotes specific RAC1, NUMB, and PKM splicing isoforms that are major triggers of colon tumorigenesis. By testing the pathway's activity in patient tumor samples, we find ELK1, MYC, and PTBP1 to be overexpressed in conjunction with oncogenic KRAS mutations, and show that these mutations increase ELK1 levels via the RAS-MAPK pathway. We thus illuminate, for the first time, a full regulatory pathway connecting prevalent cancerous mutations to functional tumor-inducing splicing aberrations. Our results demonstrate our method is applicable to different cancers to reveal regulatory pathways promoting splicing aberrations. PMID:26860615

  9. Regulation of Chemoresistance Via Alternative Messenger RNA Splicing

    PubMed Central

    Eblen, Scott T.

    2012-01-01

    The acquisition of drug resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that have were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell’s response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype. PMID:22248731

  10. Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells

    PubMed Central

    Zhang, Yanli; Kwok, Jamie Sui-Lam; Choi, Pui-Wah; Liu, Minghua; Yang, Junzheng; Singh, Margit; Ng, Shu-Kay; Welch, William R.; Muto, Michael G.; Tsui, Stephen KW; Sugrue, Stephen P.; Berkowitz, Ross S.; Ng, Shu-Wing

    2016-01-01

    Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells. PMID:26871283

  11. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  12. Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

    PubMed

    Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

    2013-01-01

    Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

  13. A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.

    PubMed

    Gao, Jun-Li; Fan, Yu-Jie; Wang, Xiu-Ye; Zhang, Yu; Pu, Jia; Li, Liang; Shao, Wei; Zhan, Shuai; Hao, Jianjiang; Xu, Yong-Zhen

    2015-04-01

    Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.

  14. Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors.

    PubMed Central

    Hanamura, A; Cáceres, J F; Mayeda, A; Franza, B R; Krainer, A R

    1998-01-01

    The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo. PMID:9630249

  15. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  16. Cloning and characterization of a Leishmania gene encoding a RNA spliced leader sequence.

    PubMed Central

    Miller, S I; Landfear, S M; Wirth, D F

    1986-01-01

    Recent studies on leishmania enriettii tubulin mRNAs revealed a 35 nucleotide addition to their 5' end. The gene that codes for this 35 nucleotide leader sequence has now been cloned and sequenced. In the Leishmania genome, the spliced leader gene exists as a tandem repeat of 438 bases. There are approximately 150 copies of this gene comprising 0.1% of the parasite genome. This gene codes for a 85 nucleotide transcript that contains the spliced leader at its 5' end. The 35 nucleotide sequence and the regions immediately 5' and 3' to it are highly conserved across trypanosomatids. We have detected a RNA molecule that is a putative by-product of the processing reaction in which the 35 nucleotide spliced leader has been transferred to mRNA. We suggest that this molecule is the remnant of the spliced leader transcript after removal of the 35 nucleotide spliced leader. Images PMID:2429261

  17. The splicing fate of plant SPO11 genes.

    PubMed

    Sprink, Thorben; Hartung, Frank

    2014-01-01

    Toward the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in Arabidopsis thaliana are essential for the initiation of double strand breaks (DSBs) during the meiotic prophase. In nearly all eukaryotic organisms DSB induction during prophase I by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO) as physical connections between the homologous chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa, and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of alternative splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non-functional as they either showed intron retention (IR) or shortened exons. However, the positional distribution and number of alternative splicing events vary strongly between the different plants. The cDNAs showed in most cases premature termination codons (PTCs) due to frameshift. Nevertheless, in some cases we found alternatively spliced but functional cDNAs. These findings let us suggest that alternative splicing of SPO11 depends on the respective gene sequence and on the plant species. Therefore, this conserved mechanism might play a role concerning regulation of SPO11.

  18. U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

    PubMed

    Park, Sung Mi; Ou, Jianhong; Chamberlain, Lynn; Simone, Tessa M; Yang, Huan; Virbasius, Ching-Man; Ali, Abdullah M; Zhu, Lihua Julie; Mukherjee, Siddhartha; Raza, Azra; Green, Michael R

    2016-05-19

    Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

  19. scaRNAs regulate splicing and vertebrate heart development.

    PubMed

    Patil, Prakash; Kibiryeva, Nataliya; Uechi, Tamayo; Marshall, Jennifer; O'Brien, James E; Artman, Michael; Kenmochi, Naoya; Bittel, Douglas C

    2015-08-01

    Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.

  20. The bromodomain protein BRD4 regulates splicing during heat shock

    PubMed Central

    Hussong, Michelle; Kaehler, Christian; Kerick, Martin; Grimm, Christina; Franz, Alexandra; Timmermann, Bernd; Welzel, Franziska; Isensee, Jörg; Hucho, Tim; Krobitsch, Sylvia; Schweiger, Michal R.

    2017-01-01

    The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies. PMID:27536004

  1. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

    PubMed Central

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-01-01

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts. PMID:23892457

  2. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

    PubMed

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-08-14

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

  3. BRR2a Affects Flowering Time via FLC Splicing

    PubMed Central

    Mahrez, Walid; Shin, Juhyun; Exner, Vivien; Siretskiy, Alexey; Köhler, Claudia

    2016-01-01

    Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects. PMID:27100965

  4. RNA splicing in a new rhabdovirus from Culex mosquitoes.

    PubMed

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-07-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

  5. [Perspectives of RNA interference application in the therapy of diseases associated with defects in alternative RNA splicing].

    PubMed

    Wysokiński, Daniel; Błasiak, Janusz

    2012-09-18

    The primary transcript of an eukaryotic gene (pre-mRNA) is composed of coding regions--exons intervened by non-coding introns--which are removed in the RNA splicing process, leading to the formation of mature, intron-free mRNA. Alternative splicing of pre-mRNA is responsible for high complexity of the cellular proteome and expresses effective use of genetic information contained in genomic DNA. Alternative splicing plays important roles in the organism, including apoptosis regulation or development and plasticity of the nervous system. The main role of alternative splicing is differential, dependent on conditions and the cell type, splicing of mRNA, generating diverse transcripts from one gene, and, after the translation, different isoforms of a particular protein. Because of the high complexity of this mechanism, alternative splicing is particularly prone to errors. The perturbations resulting from mutations in the key sequences for splicing regulations are especially harmful. The pathogenesis of numerous diseases results from disturbed alternative RNA splicing, and those include cancers and neurodegenerative disorders. The treatment of these conditions is problematic due to their genetic background and currently RNA interference, which is a common mechanism of eukaryotic gene regulation, is being studied. Initial successes in the attempts of silencing the expression of faulty protein isoforms support the idea of using RNA interference in targeting disease related to disturbances in alternative splicing of RNA.

  6. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  7. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

  8. Alternative splicing in the nervous system: an emerging source of diversity and regulation.

    PubMed

    Lee, Christopher J; Irizarry, Kris

    2003-10-15

    Alternative splicing is emerging as a major mechanism of functional regulation in the human genome. Previously considered to be an unusual event, it has been detected by many genomics studies in 40%-60% of human genes. Moreover, it appears to be of central importance for neuronal genes and other genes involved in "information processing" functions. In this review, we will summarize alternative splicing's effects on mRNA transcripts, protein products, biological function, and human disease, focusing on genes of neuropsychiatric interest. We will also describe the latest experimental methods and database resources that can help neuroscientists make use of alternative splicing in their own research.

  9. SPACE: an algorithm to predict and quantify alternatively spliced isoforms using microarrays

    PubMed Central

    Anton, Miguel A; Gorostiaga, Dorleta; Guruceaga, Elizabeth; Segura, Victor; Carmona-Saez, Pedro; Pascual-Montano, Alberto; Pio, Ruben; Montuenga, Luis M; Rubio, Angel

    2008-01-01

    Exon and exon+junction microarrays are promising tools for studying alternative splicing. Current analytical tools applied to these arrays lack two relevant features: the ability to predict unknown spliced forms and the ability to quantify the concentration of known and unknown isoforms. SPACE is an algorithm that has been developed to (1) estimate the number of different transcripts expressed under several conditions, (2) predict the precursor mRNA splicing structure and (3) quantify the transcript concentrations including unknown forms. The results presented here show its robustness and accuracy for real and simulated data. PMID:18312629

  10. An NS-segment exonic splicing enhancer regulates influenza A virus replication in mammalian cells

    PubMed Central

    Huang, Xiaofeng; Zheng, Min; Wang, Pui; Mok, Bobo Wing-Yee; Liu, Siwen; Lau, Siu-Ying; Chen, Pin; Liu, Yen-Chin; Liu, Honglian; Chen, Yixin; Song, Wenjun; Yuen, Kwok-Yung; Chen, Honglin

    2017-01-01

    Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. Through genetic analysis and functional characterization, we here show that the NS segment of H7N9 virus contains a unique G540A substitution, located within a previously undefined exonic splicing enhancer (ESE) motif present in the NEP mRNA of influenza A viruses. G540A supports virus replication in mammalian cells while retaining replication ability in avian cells. Host splicing regulator, SF2, interacts with this ESE to regulate splicing of NEP/NS1 mRNA and G540A substitution affects SF2–ESE interaction. The NS1 protein directly interacts with SF2 in the nucleus and modulates splicing of NS mRNAs during virus replication. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells. PMID:28323816

  11. Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.

    PubMed

    Derelle, Romain; Momose, Tsuyoshi; Manuel, Michael; Da Silva, Corinne; Wincker, Patrick; Houliston, Evelyn

    2010-04-01

    Replacement of mRNA 5' UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3' of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare.

  12. Expression of intron-containing C. elegans heat shock genes in mouse cells demonstrates divergence of 3' splice site recognition sequences between nematodes and vertebrates, and an inhibitory effect of heat shock on the mammalian splicing apparatus.

    PubMed Central

    Kay, R J; Russnak, R H; Jones, D; Mathias, C; Candido, E P

    1987-01-01

    Splicing of a pair of intron-containing heat shock genes from Caenorhabditis elegans has been studied in transfected mouse cells. The hsp16-1 and hsp16-48 genes of C. elegans encode 16,000 Da heat shock polypeptides. Each gene contains a short intron of 52 (hsp16-1) or 55 (hsp16-48) base pairs. When these genes were introduced into mouse cells, they were efficiently induced following heat shock, but splicing of the introns was abnormal. In mouse cells, cleavage of the hsp16 transcripts occurred at the correct 5' splice sites, but the 3' splice sites were located at AG dinucleotides downstream of the correct sites. This aberrant splicing was not solely due to the small size of the C. elegans introns, since a hsp16-1 gene containing an intron enlarged by tandem duplication showed exactly the same splicing pattern. The mouse cells thus seem to be unable to recognize the natural 3' splice sites of the C. elegans transcripts. The efficiency of splicing was greatly reduced under heat shock conditions, and unspliced transcripts accumulated in the nucleus. During a subsequent recovery period at 37 degrees C, these transcripts were spliced and transported to the cytoplasm. Images PMID:3588308

  13. In vivo translation and stability of trans-spliced mRNAs in nematode embryos.

    PubMed

    Cheng, Guofeng; Cohen, Leah; Mikhli, Claudette; Jankowska-Anyszka, Marzena; Stepinski, Janusz; Darzynkiewicz, Edward; Davis, Richard E

    2007-06-01

    Spliced leader trans-splicing adds a short exon, the spliced leader (SL), to pre-mRNAs to generate 5' ends of mRNAs. Addition of the SL in metazoa also adds a new cap to the mRNA, a trimethylguanosine (m(3)(2,2,7)GpppN) (TMG) that replaces the typical eukaryotic monomethylguanosine (m7GpppN)(m7G) cap. Both trans-spliced (m3(2,2,7)GpppN-SL-RNA) and not trans-spliced (m7GpppN-RNA) mRNAs are present in the same cells. Previous studies using cell-free systems to compare the overall translation of trans-spliced versus non-trans-spliced RNAs led to different conclusions. Here, we examine the contribution of m3(2,2,7)GpppG-cap and SL sequence and other RNA elements to in vivo mRNA translation and stability in nematode embryos. Although 70-90% of all nematode mRNAs have a TMG-cap, the TMG cap does not support translation as well as an m7G-cap. However, when the TMG cap and SL are present together, they synergistically interact and translation is enhanced, indicating both trans-spliced elements are necessary to promote efficient translation. The SL by itself does not act as a cap-independent enhancer of translation. The poly(A)-tail synergistically interacts with the mRNA cap enhancing translation and plays a greater role in facilitating translation of TMG-SL mRNAs. In general, recipient mRNA sequences between the SL and AUG and the 3' UTR do not significantly contribute to the translation of trans-spliced mRNAs. Overall, the combination of TMG cap and SL contribute to mRNA translation and stability in a manner typical of a eukaryotic m7G-cap and 5' UTRs, but they do not differentially enhance mRNA translation or stability compared to RNAs without the trans-spliced elements.

  14. Alternative splicing in chronic myeloid leukemia (CML): a novel therapeutic target?

    PubMed

    Adamia, Sophia; Pilarski, Patrick M; Bar-Natan, Michal; Stone, Richard M; Griffin, James D

    2013-09-01

    Although the imatinib based therapy of chronic myeloid leukemia (CML) represents a triumph of medicine, not all patients with CML benefit from this drug due to the development of resistance and intolerance. The interruption of imatinib treatment is often followed by clinical relapse, suggesting a failure in the killing of residual leukaemic stem cells. There is need to identify alternative selective molecular targets for this disease and develop more effective therapeutic approaches. Alternative pre-mRNA splicing (AS) is an epigenetic process that greatly diversifies the repertoire of the transcriptome. AS orchestrates interactions between various types of proteins and between proteins and nucleic acids. Changes caused by individual splicing events in the cells are small, however, "splicing programs" typically react to these individual changes with considerable effects in cell proliferation, cell survival, and apoptosis. Current evidence suggests a pivotal role of AS in leukemias, particularly in myelodisplastic syndrome (MDS) and chronic lymphocyte leukemia (CLL). From these studies and studies in other malignances, it is clear that splicing abnormalities play a significant role in malignant transformation. Evaluation of AS events in CML can be used to identify novel disease markers and drugsensitive targets to overcome the limits of the small molecule inhibitors currently used for treating patients with CML. The use of aberrant splice variants as disease markers has been reported, however, little is known about the use of splicing abnormalities as drug targets in CML. Herein we discuss potential therapeutic approaches that can be used to target splicing abnormalities in CML.

  15. Splice junction mutation in some Ashkenazi Jews with Tay-Sachs disease: Evidence against a single defect within this ethnic group

    SciTech Connect

    Myerowitz, R. )

    1988-06-01

    Tay-Sachs disease is an inherited disorder in which the {alpha} chain of the lysosomal enzyme {beta}-N-acetylhexosaminidase A bears the mutation. Ashkenazi Jews are found to be carriers for a severe type of Tay-Sachs disease, the classic form, 10 times more frequently than the general population. Ashkenazi Jewish patients with classic Tay-Sachs disease have appeared to be clinically and biochemically identical, and the usual assumption has been that they harbor the same {alpha}-chain mutation. The author has isolated the {alpha}-chain gene from an Ashkenazi Jewish patient, GM2968, with classic Tay-Sachs disease and compared its nucleotide sequences with that of the normal {alpha}-chain gene in the promoter region, exon and splice junction regions, and polyadenylylation signal area. Only one difference was observed between these sequences. The alteration is presumed to be functionally significant and to result in aberrant mRNA splicing. Utilizing the polymerase chain reaction to amplify the region encompassing the mutation, the author developed an assay to screen patients and heterozygote carriers for this mutation. Surprisingly, in each of two Ashkenazi patients, only one {alpha}-chain allele harbored the splice junction mutation. Only one parent of each of these patients was positive for the defect. Another Ashkenazi patient did not bear this mutation at all nor did either of the subject's parents. The data are consistent with the presence of more than one mutation underlying the classic form of Tay-Sachs disease in the Ashkenazi Jewish population.

  16. NF1 mutation rather than individual genetic variability is the main determinant of the NF1-transcriptional profile of mutations affecting splicing.

    PubMed

    Pros, Eva; Larriba, Sara; López, Eva; Ravella, Anna; Gili, M Lluïsa; Kruyer, Helena; Valls, Joan; Serra, Eduard; Lázaro, Conxi

    2006-11-01

    A significant number of neurofibromatosis type 1 (NF1) mutations result in exon skipping. The majority of these mutations do not occur in the canonical splice sites and can produce different aberrant transcripts whose proportions have not been well studied. It has been hypothesized that differences in the mutation-determined NF1-transcriptional profile could partially explain disease variability among patients bearing the same NF1 splice defect. In order to gain insight into these aspects, we analyzed the proportion of the different transcripts generated by nine NF1-splicing mutations in 30 patients. We assessed the influence of the mutation in the NF1-related transcriptional profiles and investigated the existence of individual differences in a global manner. We analyzed potential differences in tissue-specific transcriptional profiles and evaluated the influence of sample processing and mRNA nonsense-mediated decay (NMD). Small transcriptional differences were found in neurofibromas and neurofibroma-derived Schwann cells (SC) compared to blood. We also detected a higher cell culture-dependent NMD. We observed that mutation per se explains 93.5% of the profile variability among mutations studied. However, despite the importance of mutation in determining the proportion of NF1 transcripts generated, we found certain variability among patients with the same mutation. From our results, it seems that genetic factors influencing RNA processing play a minor role in determining the NF1-transcriptional profile. Nevertheless neurofibromin studies would clarify whether these small differences translate into significant functional changes that could explain the great clinical expressivity observed in the disease or any of the disease-related traits.

  17. Cellular stress and RNA splicing.

    PubMed

    Biamonti, Giuseppe; Caceres, Javier F

    2009-03-01

    In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

  18. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.

  19. Splicing of aged fibers

    NASA Astrophysics Data System (ADS)

    Volotinen, Tarja T.; Yuce, Hakan H.; Bonanno, Nicholas; Frantz, Rolf A.; Duffy, Sean

    1993-11-01

    The deployment of fiber in the subscriber loop will require that an optical fiber network maintain the highest possible level of reliability over time, despite being subjected to extremes of temperature, humidity, and other environmental and mechanical stresses imposed on the outside plant. At the same time, both the initial cost and the ongoing maintenance expenses for loop equipment must be kept low. Fiber in the Loop (FITL) applications will entail increased fiber handling. Cable lengths will be shorter, and fiber counts higher, than has been the case so far in long-distance applications. There will also be more cable sheath openings per unit length of cable and/or fiber, as well as more splicing and connectorization. It may become a common practice that a customer is connected to a cable installed many years earlier. In subscriber loops, cables and fibers will be installed in harsher and more varying environments. Fibers will be exposed to higher humidity and temperature, particularly in splice boxes mounted on building walls, in pedestal cabinets, and in other similar enclosures. Corrosive gases and/or liquids may also be present at some locations and will adversely affect the fibers. The combination of increased handling, greater exposure, and more stressful environments may give rise to a need for new, more stringent requirements for fiber mechanical reliability. These can include increaSed fiber strength, increased aging resistance, and increased fatigue resistance.

  20. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    PubMed

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcáRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

  1. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  2. Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.

    PubMed

    Schor, Ignacio E; Llères, David; Risso, Guillermo J; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I; Kornblihtt, Alberto R

    2012-01-01

    Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin

  3. Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

    PubMed Central

    Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

    2012-01-01

    Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin

  4. Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing.

    PubMed

    Alberstein, Moti; Amit, Maayan; Vaknin, Keren; O'Donnell, Amanda; Farhy, Chen; Lerenthal, Yaniv; Shomron, Noam; Shaham, Ohad; Sharrocks, Andrew D; Ashery-Padan, Ruth; Ast, Gil

    2007-11-01

    Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.

  5. SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs.

    PubMed

    Ma, Xiaoju Max; Yoon, Sang-Oh; Richardson, Celeste J; Jülich, Kristina; Blenis, John

    2008-04-18

    Different protein complexes form on newly spliced mRNA to ensure the accuracy and efficiency of eukaryotic gene expression. For example, the exon junction complex (EJC) plays an important role in mRNA surveillance. The EJC also influences the first, or pioneer round of protein synthesis through a mechanism that is poorly understood. We show that the nutrient-, stress-, and energy-sensing checkpoint kinase, mTOR, contributes to the observed enhanced translation efficiency of spliced over nonspliced mRNAs. We demonstrate that, when activated, S6K1 is recruited to the newly synthesized mRNA by SKAR, which is deposited at the EJC during splicing, and that SKAR and S6K1 increase the translation efficiency of spliced mRNA. Thus, SKAR-mediated recruitment of activated S6K1 to newly processed mRNPs serves as a conduit between mTOR checkpoint signaling and the pioneer round of translation when cells exist in conditions supportive of protein synthesis.

  6. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    SciTech Connect

    Giblin, P.; Kavathas, P. ); Ledbetter, J.A. )

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  7. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

    PubMed Central

    An, Ping; Burge, Christopher B

    2005-01-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859

  8. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  9. MORPHEUS' MOLECULE1 is required to prevent aberrant RNA transcriptional read-through in Arabidopsis.

    PubMed

    Zhou, Yue; Zhang, Jun; Lin, Huixin; Guo, Guangqin; Guo, Yan

    2010-11-01

    Several pathways function to remove aberrant mRNA in eukaryotic cells; however, the exact mechanisms underlying the restriction of aberrant mRNA transcription are poorly understood. In this study, we found that MORPHEUS' MOLECULE1 (MOM1) is a key component of this regulatory machinery. The Arabidopsis (Arabidopsis thaliana) mom1-44 mutation was identified by luciferase imaging in transgenic plants harboring a cauliflower mosaic virus 35S promoter-LUCIFERASE transgene lacking the 3'-untranslated region. In the mom1-44 mutant, transcriptional read-though occurred in genes with an aberrant RNA structure. Analysis of an RNA-dependent RNA polymerase2 mom1 double mutant revealed that the RNA-directed DNA methylation pathway is not involved in this regulatory process. Moreover, the prevention of aberrant mRNA transcriptional read-through by MOM1 is gene locus and transgene copy number independent.

  10. Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

    PubMed

    Ág, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsébet

    2015-12-01

    Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

  11. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

    SciTech Connect

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A.; Khoder, Mamdouh I.; Shamy, Magdy; Muñoz, Manuel J.; and others

    2015-07-15

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.

  12. Myocardial alternative RNA splicing and gene expression profiling in early stage hypoplastic left heart syndrome.

    PubMed

    Ricci, Marco; Xu, Yanji; Hammond, Harriet L; Willoughby, David A; Nathanson, Lubov; Rodriguez, Maria M; Vatta, Matteo; Lipshultz, Steven E; Lincoln, Joy

    2012-01-01

    Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome.

  13. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

    PubMed

    Treisman, R; Proudfoot, N J; Shander, M; Maniatis, T

    1982-07-01

    We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

  14. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

    PubMed

    Treisman, R; Orkin, S H; Maniatis, T

    1983-04-14

    Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

  15. Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

    PubMed

    Schimmer, Bernard P; Cordova, Martha

    2015-06-15

    The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

  16. A new view of transcriptome complexity and regulation through the lens of local splicing variations

    PubMed Central

    Vaquero-Garcia, Jorge; Barrera, Alejandro; Gazzara, Matthew R; González-Vallinas, Juan; Lahens, Nicholas F; Hogenesch, John B; Lynch, Kristen W; Barash, Yoseph

    2016-01-01

    Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease. DOI: http://dx.doi.org/10.7554/eLife.11752.001 PMID:26829591

  17. The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

    PubMed Central

    Malone, Colin D.; Mestdagh, Claire; Akhtar, Junaid; Kreim, Nastasja; Deinhard, Pia; Sachidanandam, Ravi; Treisman, Jessica

    2014-01-01

    The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. PMID:25104425

  18. Splicing of intron 3 of human BACE requires the flanking introns 2 and 4.

    PubMed

    Annies, Maik; Stefani, Muriel; Hueber, Andreas; Fischer, Frauke; Paganetti, Paolo

    2009-10-16

    Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer's disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.

  19. Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

    PubMed Central

    Struck, Adam J.; Gupta, Riti; Farnsworth, Dylan R.; Mahady, Amy E.; Eichinger, Katy; Thornton, Charles A.; Wang, Eric T.; Berglund, J. Andrew

    2016-01-01

    Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM. PMID:27681373

  20. Detection of circulating prostate tumor cells: alternative spliced variant of PSM induced false-positive result.

    PubMed

    Hisatomi, Hisashi; Nagao, Kumi; Kawakita, Mutsuji; Matsuda, Tadashi; Hirata, Hiroyuki; Yamamoto, Shigeki; Nakamoto, Takaaki; Harasawa, Hiroshi; Kaneko, Noboru; Hikiji, Kazumasa; Tsukada, Yutaka

    2002-11-01

    RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.

  1. Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

    PubMed

    Wang, Xiu-Ye; Zheng, Zeng-Zhang; Song, Hong-Sheng; Xu, Yong-Zhen

    2014-01-01

    Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5' splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5' splice site at intron 3 of Lepidopteran dsx. We

  2. Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

    PubMed Central

    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

  3. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

    PubMed

    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  4. Quantification of co-transcriptional splicing from RNA-Seq data.

    PubMed

    Herzel, Lydia; Neugebauer, Karla M

    2015-09-01

    During gene expression, protein-coding transcripts are shaped by multiple processing events: 5' end capping, pre-mRNA splicing, RNA editing, and 3' end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3' end cleavage or post-transcriptionally. Pre-mRNA splicing - the removal of introns and ligation of exons - can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.

  5. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing.

  6. Alternative splicing may be responsible for heterogeneity of thyroglobulin structure.

    PubMed

    Mercken, L; Simons, M J; Brocas, H; Vassart, G

    1989-02-01

    During the cloning of the bovine thyroglobulin cDNA, the restriction map of one of the recombinant plasmids was in disagreement with that of the full-length double-stranded thyroglobulin cDNA. When compared to the bovine Tg mRNA sequence, this cDNA clone exhibits a 333-nucleotide deletion which corresponds precisely to 2 exons of the Tg gene. It is thus likely that alternative processing of the premessenger RNA is at the origin of the deletion. The presence of giant introns in the vicinity of the dispensable exons may also reflect some error level in the splicing mechanism. Together with previous results the alternative splicing described in this study indicates that alternative processing of the Tg transcripts may be at the origin of thyroglobulin isoforms.

  7. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    PubMed

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  8. Aberration correction of unstable resonators

    NASA Technical Reports Server (NTRS)

    Lang, Robert J. (Inventor)

    1994-01-01

    Construction of aspheric reflectors for unstable resonator lasers to provide an arbitrary laser mode inside the resonator to correct aberrations of an output beam by the construction of the shape of an end reflector opposite the output reflector of the resonator cavity, such as aberrations resulting from refraction of a beam exiting the solid of the resonator having an index of refraction greater than 1 or to produce an aberration in the output beam that will precisely compensate for the aberration of an optical train into which the resonator beam is coupled.

  9. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

    PubMed

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

  10. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

    PubMed Central

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing. PMID:26546116

  11. Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

    PubMed Central

    Zhang, Yan; Jiang, Qiang; Huang, Jinming; Ju, Zhihua; Wang, Xiuge; Zhong, Jifeng; Wang, Changfa

    2016-01-01

    Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378. PMID:27669152

  12. Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

    PubMed Central

    Metherall, J E; Collins, F S; Pan, J; Weissman, S M; Forget, B G

    1986-01-01

    A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3780671

  13. SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

    PubMed Central

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  14. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

  15. Epithelial splicing regulatory protein 1 and 2 paralogues correlate with splice signatures and favorable outcome in human colorectal cancer

    PubMed Central

    Deloria, Abigail J.; Höflmayer, Doris; Kienzl, Philip; Łopatecka, Justyna; Sampl, Sandra; Klimpfinger, Martin; Braunschmid, Tamara; Bastian, Fabienne; Lu, Lingeng; Marian, Brigitte; Stättner, Stefan; Holzmann, Klaus

    2016-01-01

    ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression. Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P=0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P=0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15–0.91, P=0.032; ESRP2: HR=0.23, 95%CI 0.08–0.65, P=0.006). Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC. PMID:27650542

  16. Tissue Restricted Splice Junctions Originate Not Only from Tissue-Specific Gene Loci, but Gene Loci with a Broad Pattern of Expression

    PubMed Central

    Hestand, Matthew S.; Zeng, Zheng; Coleman, Stephen J.; Liu, Jinze; MacLeod, James N.

    2015-01-01

    Cellular mechanisms that achieve protein diversity in eukaryotes are multifaceted, including transcriptional components such as RNA splicing. Through alternative splicing, a single protein-coding gene can generate multiple mRNA transcripts and protein isoforms, some of which are tissue-specific. We have conducted qualitative and quantitative analyses of the Bodymap 2.0 messenger RNA-sequencing data from 16 human tissue samples and identified 209,363 splice junctions. Of these, 22,231 (10.6%) were not previously annotated and 21,650 (10.3%) were expressed in a tissue-restricted pattern. Tissue-restricted alternative splicing was found to be widespread, with approximately 65% of expressed multi-exon genes containing at least one tissue-specific splice junction. Interestingly, we observed many tissue-specific splice junctions not only in genes expressed in one or a few tissues, but also from gene loci with a broad pattern of expression. PMID:26713731

  17. Innovations in Proteomic Profiling of Cancers: Alternative Splice Variants as a New Class of Cancer Biomarker Candidates and Bridging of Proteomics with Structural Biology

    PubMed Central

    Omenn, Gilbert S.; Menon, Rajasree; Zhang, Yang

    2013-01-01

    Alternative splicing allows a single gene to generate multiple RNA transcripts which can be translated into functionally diverse protein isoforms. Current knowledge of splicing is derived mainly from RNA transcripts, with very little known about the expression level, 3D structures, and functional differences of the proteins. Splicing is a remarkable phenomenon of molecular and biological evolution. Studies which simply report up-regulation or down-regulation of protein or mRNA expression are confounded by the effects of mixtures of these isoforms. Besides understanding the net biological effects of the mixtures, we may be able to develop biomarker tests based on the observable differential expression of particular splice variants or combinations of splice variants in specific disease states. Here we review our work on differential expression of splice variant proteins in cancers and the feasibility of integrating proteomic analysis with structure-based conformational predictions of the differences between such isoforms. PMID:23603631

  18. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  19. Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse.

    PubMed

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew J A

    2010-04-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy.

  20. Camera processing with chromatic aberration.

    PubMed

    Korneliussen, Jan Tore; Hirakawa, Keigo

    2014-10-01

    Since the refractive index of materials commonly used for lens depends on the wavelengths of light, practical camera optics fail to converge light to a single point on an image plane. Known as chromatic aberration, this phenomenon distorts image details by introducing magnification error, defocus blur, and color fringes. Though achromatic and apochromatic lens designs reduce chromatic aberration to a degree, they are complex and expensive and they do not offer a perfect correction. In this paper, we propose a new postcapture processing scheme designed to overcome these problems computationally. Specifically, the proposed solution is comprised of chromatic aberration-tolerant demosaicking algorithm and post-demosaicking chromatic aberration correction. Experiments with simulated and real sensor data verify that the chromatic aberration is effectively corrected.

  1. Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing.

    PubMed

    McPheeters, D S; Schwer, B; Muhlenkamp, P

    2000-03-15

    Using site-specific incorporation of the photo-chemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3' splice sites. One of these is an unidentified approximately 122 kDa protein that cross-links to 3' splice sites during formation of the pre--spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3' splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3' splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3' splice site of yeast introns is contacted much earlier than previously envisioned by a protein of approximately 120 kDa, and that a transient association of Prp22p with the 3' splice site occurs between the first and second catalytic steps.

  2. A computational and experimental approach toward a priori identification of alternatively spliced exons

    PubMed Central

    PHILIPPS, DANA L.; PARK, JUNG W.; GRAVELEY, BRENTON R.

    2004-01-01

    Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data. PMID:15525709

  3. Antisense suppression of donor splice site mutations in the dystrophin gene transcript

    PubMed Central

    Fletcher, Sue; Meloni, Penny L; Johnsen, Russell D; Wong, Brenda L; Muntoni, Francesco; Wilton, Stephen D

    2013-01-01

    We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis. PMID:24498612

  4. Splicing inhibition decreases phosphorylation level of Ser2 in Pol II CTD

    PubMed Central

    Koga, Mitsunori; Hayashi, Megumi; Kaida, Daisuke

    2015-01-01

    Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5′ end capping, splicing and 3′ end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms. PMID:26202968

  5. Emerging functions of SRSF1, splicing factor and oncoprotein, in RNA metabolism and cancer.

    PubMed

    Das, Shipra; Krainer, Adrian R

    2014-09-01

    Serine/Arginine Splicing Factor 1 (SRSF1) is the archetype member of the SR protein family of splicing regulators. Since its discovery over two decades ago, SRSF1 has been repeatedly surprising and intriguing investigators by the plethora of complex biologic pathways it regulates. These include several key aspects of mRNA metabolism, such as mRNA splicing, stability, and translation, as well as other mRNA-independent processes, such as miRNA processing, protein sumoylation, and the nucleolar stress response. In this review, the structural features of SRSF1 are discussed as they relate to the intricate mechanism of splicing and the multiplicity of functions it performs. Similarly, a list of relevant alternatively spliced transcripts and SRSF1 interacting proteins is provided. Finally, emphasis is given to the deleterious consequences of overexpression of the SRSF1 proto-oncogene in human cancers, and the complex mechanisms and pathways underlying SRSF1-mediated transformation. The accumulated knowledge about SRSF1 provides critical insight into the integral role it plays in maintaining cellular homeostasis and suggests new targets for anticancer therapy. Mol Cancer Res; 12(9); 1195-204. ©2014 AACR.

  6. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

    SciTech Connect

    Tian, Ping-Ge; Jiang, Zhi-Xin; Li, Jian-Hua; Zhou, Zhe; Zhang, Qing-Hua

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  7. Mesenchymal stem cell contact promotes CCN1 splicing and transcription in myeloma cells.

    PubMed

    Dotterweich, Julia; Ebert, Regina; Kraus, Sabrina; Tower, Robert J; Jakob, Franz; Schütze, Norbert

    2014-06-25

    CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.

  8. Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

    PubMed Central

    Gantla, S; Bakker, C T; Deocharan, B; Thummala, N R; Zweiner, J; Sinaasappel, M; Roy Chowdhury, J; Bosma, P J; Roy Chowdhury, N

    1998-01-01

    Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression. PMID:9497253

  9. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

    PubMed

    Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

    2014-04-01

    Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

  10. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    PubMed Central

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  11. Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster.

    PubMed

    May, Gemma E; Olson, Sara; McManus, C Joel; Graveley, Brenton R

    2011-02-01

    Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.

  12. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.

    PubMed

    Hall, Megan P; Nagel, Roland J; Fagg, W Samuel; Shiue, Lily; Cline, Melissa S; Perriman, Rhonda J; Donohue, John Paul; Ares, Manuel

    2013-05-01

    Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

  13. Chromosome Aberrations in Astronauts

    NASA Technical Reports Server (NTRS)

    George, Kerry A.; Durante, M.; Cucinotta, Francis A.

    2007-01-01

    A review of currently available data on in vivo induced chromosome damage in the blood lymphocytes of astronauts proves that, after protracted exposure of a few months or more to space radiation, cytogenetic biodosimetry analyses of blood collected within a week or two of return from space provides a reliable estimate of equivalent radiation dose and risk. Recent studies indicate that biodosimetry estimates from single spaceflights lie within the range expected from physical dosimetry and biophysical models, but very large uncertainties are associated with single individual measurements and the total sample population remains low. Retrospective doses may be more difficult to estimate because of the fairly rapid time-dependent loss of "stable" aberrations in blood lymphocytes. Also, biodosimetry estimates from individuals who participate in multiple missions, or very long (interplanetary) missions, may be complicated by an adaptive response to space radiation and/or changes in lymphocyte survival and repopulation. A discussion of published data is presented and specific issues related to space radiation biodosimetry protocols are discussed.

  14. HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome

    PubMed Central

    Baeyens, Ann; Naessens, Evelien; Van Nuffel, Anouk; Weening, Karin E.; Reilly, Anne-Marie; Claeys, Eva; Trypsteen, Wim; Vandekerckhove, Linos; Eyckerman, Sven; Gevaert, Kris; Verhasselt, Bruno

    2016-01-01

    To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions. PMID:27721439

  15. A novel donor splice-site mutation of major intrinsic protein gene associated with congenital cataract in a Chinese family

    PubMed Central

    Zeng, Lu; Liu, Wenqiang; Feng, Wenguo; Wang, Xing; Dang, Hui; Gao, Luna; Yao, Jing

    2013-01-01

    Purpose To identify the disease-causing gene in a Chinese family with autosomal dominant congenital cataract. Methods Clinical and ophthalmologic examinations were performed on all members of a Chinese family with congenital cataract. Nine genes associated with congenital cataract were screened using direct DNA sequencing. Mutations were confirmed using restriction fragment length polymorphism (RFLP) analysis. The mutated major intrinsic protein (MIP) minigene, which carries the disease-causing splice-site mutation, and the wild-type (WT) MIP minigene were constructed using the pcDNA3.1 expression vector. Wild-type and mutant MIP minigene constructs were transiently transfected into HeLa cells. After 48 h of incubation at 37 °C, total RNA isolation and reverse transcription (RT)–PCR analysis were performed, and PCR products were separated and confirmed with sequencing. Results Direct DNA sequence analysis identified a novel splice-site mutation in intron 3 (c.606+1 G>A) of the MIP gene. To investigate the manner in which the splice donor mutation could affect mRNA splicing, WT and mutant MIP minigenes were inserted in the pcDNA3.1 (+) vector. Constructs were transfected into HeLa cells. RT–PCR analysis showed that the donor splice site mutation led to deletion of exon 3 in the mRNA encoded by the MIP gene. Conclusions The present study identified a novel donor splice-site mutation (c.606+1G>A) in the MIP gene in a Chinese family with congenital cataract. In vitro RT–PCR analysis showed that this splice-site mutation resulted in the deletion of exon 3 from mRNA encoded by the MIP gene. This is the first report to show that donor splice-site mutation in MIP gene can cause autosomal dominant congenital cataract. PMID:24319327

  16. MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Oma, Yoko; Onishi, Hayato; Nezu, Yuriko; Sasagawa, Noboru; Nukina, Nobuyuki; Ishiura, Shoichi

    2009-10-01

    The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

  17. Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin

    PubMed Central

    Trejbalová, Kateřina; Blažková, Jana; Matoušková, Magda; Kučerová, Dana; Pecnová, Lubomíra; Vernerová, Zdenka; Heráček, Jiří; Hirsch, Ivan; Hejnar, Jiří

    2011-01-01

    Syncytin-1 and -2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. We studied the epigenetic suppression of ERVWE1 and ERVFRDE1 5′-long terminal repeats by DNA methylation and chromatin modifications. Immunoprecipitation of the provirus-associated chromatin revealed the H3K9 trimethylation at transcriptionally inactivated syncytins in HeLa cells. qRT-PCR analysis of non-spliced ERVWE1 and ERVFRDE1 mRNAs and respective env mRNAs detected efficient splicing of endogenously expressed RNAs in trophoblastic but not in non-placental cells. Pointing to the pathogenic potential of aberrantly expressed syncytin-1, we have found deregulation of transcription and splicing of the ERVWE1 in biopsies of testicular seminomas. Finally, ectopic expression experiments suggest the importance of proper chromatin context for the ERVWE1 splicing. Our results thus demonstrate that cell-specific retroviral splicing represents an additional epigenetic level controling the expression of endogenous retroviruses. PMID:21771862

  18. A new type of mutation causes a splicing defect in ATM.

    PubMed

    Pagani, Franco; Buratti, Emanuele; Stuani, Cristiana; Bendix, Regina; Dörk, Thilo; Baralle, Francisco E

    2002-04-01

    Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have identified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5' and 3' ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

  19. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  20. Evolutionary conservation of alternative splicing in chicken

    PubMed Central

    Katyal, S.; Gao, Z.; Liu, R.-Z.; Godbout, R.

    2013-01-01

    Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

  1. Primary structure of rat plasma membrane Ca(2+)-ATPase isoform 4 and analysis of alternative splicing patterns at splice site A.

    PubMed Central

    Keeton, T P; Shull, G E

    1995-01-01

    We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5'-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids. Images Figure 1 Figure 5 Figure 6 PMID:7702574

  2. Correction of Distributed Optical Aberrations

    SciTech Connect

    Baker, K; Olivier, S; Carrano, C; Phillion, D

    2006-02-12

    The objective of this project was to demonstrate the use of multiple distributed deformable mirrors (DMs) to improve the performance of optical systems with distributed aberrations. This concept is expected to provide dramatic improvement in the optical performance of systems in applications where the aberrations are distributed along the optical path or within the instrument itself. Our approach used multiple actuated DMs distributed to match the aberration distribution. The project developed the algorithms necessary to determine the required corrections and simulate the performance of these multiple DM systems.

  3. Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

    DTIC Science & Technology

    2013-07-01

    Stanchina lab, new baselines for tumor growth were derived in 8-10 weeks old athymic female mice, which were subcutaneously inoculated with 10X106 MDA-MB...the left ventricle) or orthotopically into axillary mammary fat pads of 6-week old female athymic nude mice are underway in the de Stanchina lab...splicing and the addition of a 5’ cap and a 3’ polyadenylation sequence [15]. The choice and usage of splice sites in the pre- mRNA can be

  4. RNA splicing regulates the temporal order of TNF-induced gene expression.

    PubMed

    Hao, Shengli; Baltimore, David

    2013-07-16

    When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

  5. Conserved RNA secondary structures promote alternative splicing.

    PubMed

    Shepard, Peter J; Hertel, Klemens J

    2008-08-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site strength, splicing regulators, the exon/intron architecture, and the process of pre-mRNA synthesis itself. RNA secondary structures have also been proposed to influence alternative splicing as stable RNA secondary structures that mask splice sites are expected to interfere with splice-site recognition. Using structural and functional conservation, we identified RNA structure elements within the human genome that associate with alternative splice-site selection. Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease.

  6. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

  7. An integrative characterization of recurrent molecular aberrations in glioblastoma genomes.

    PubMed

    Sintupisut, Nardnisa; Liu, Pei-Ling; Yeang, Chen-Hsiang

    2013-10-01

    Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Decades of investigations and the recent effort of the Cancer Genome Atlas (TCGA) project have mapped many molecular alterations in GBM cells. Alterations on DNAs may dysregulate gene expressions and drive malignancy of tumors. It is thus important to uncover causal and statistical dependency between 'effector' molecular aberrations and 'target' gene expressions in GBMs. A rich collection of prior studies attempted to combine copy number variation (CNV) and mRNA expression data. However, systematic methods to integrate multiple types of cancer genomic data-gene mutations, single nucleotide polymorphisms, CNVs, DNA methylations, mRNA and microRNA expressions and clinical information-are relatively scarce. We proposed an algorithm to build 'association modules' linking effector molecular aberrations and target gene expressions and applied the module-finding algorithm to the integrated TCGA GBM data sets. The inferred association modules were validated by six tests using external information and datasets of central nervous system tumors: (i) indication of prognostic effects among patients; (ii) coherence of target gene expressions; (iii) retention of effector-target associations in external data sets; (iv) recurrence of effector molecular aberrations in GBM; (v) functional enrichment of target genes; and (vi) co-citations between effectors and targets. Modules associated with well-known molecular aberrations of GBM-such as chromosome 7 amplifications, chromosome 10 deletions, EGFR and NF1 mutations-passed the majority of the validation tests. Furthermore, several modules associated with less well-reported molecular aberrations-such as chromosome 11 CNVs, CD40, PLXNB1 and GSTM1 methylations, and mir-21 expressions-were also validated by external information. In particular, modules constituting trans-acting effects with chromosome 11 CNVs and cis-acting effects with chromosome

  8. STABILIZED1 Modulates Pre-mRNA Splicing for Thermotolerance1[OPEN

    PubMed Central

    Cho, Young-Hee

    2017-01-01

    High-temperature stress often leads to differential RNA splicing, thus accumulating different types and/or amounts of mature mRNAs in eukaryotic cells. However, regulatory mechanisms underlying plant precursor mRNA (pre-mRNA) splicing in the environmental stress conditions remain elusive. Herein, we describe that a U5-snRNP-interacting protein homolog STABILIZED1 (STA1) has pre-mRNA splicing activity for heat-inducible transcripts including HEAT STRESS TRANSCRIPTION FACTORs and various HEAT SHOCK PROTEINs for the establishment of heat stress tolerance in Arabidopsis (Arabidopsis thaliana). Our cell-based splicing reporter assay demonstrated STA1 acts on pre-mRNA splicing for specific subsets of stress-related genes. Cellular reconstitution of heat-inducible transcription cascades supported the view that STA1-dependent pre-mRNA splicing plays a role in DREB2A-dependent HSFA3 expression for heat-responsive gene expression. Further genetic analysis with a loss-of-function mutant sta1-1, STA1-expressing transgenic plants in Col background, and STA1-expressing transgenic plants in the sta1-1 background verified that STA1 is essential in expression of necessary genes including HSFA3 for two-step heat stress tolerance in plants. However, constitutive overexpression of the cDNA version of HSFA3 in the sta1-1 background is unable to execute plant heat stress tolerance in sta1-1. Consistently our global target analysis of STA1 showed that its splicing activity modulates a rather broad range of gene expression in response to heat treatment. The findings of this study reveal that heat-inducible STA1 activity for pre-mRNA splicing serves as a molecular regulatory mechanism underlying the plant stress tolerance to high-temperature stress. PMID:28223317

  9. Male-specific splicing of the silkworm Imp gene is maintained by an autoregulatory mechanism.

    PubMed

    Suzuki, Masataka G; Kobayashi, Sayaka; Aoki, Fugaku

    2014-02-01

    Sexual differentiation in the silkworm Bombyx mori is controlled by sex-specific splicing of Bmdsx, in which exons 3 and 4 are skipped in males. B. mori insulin-like growth factor II mRNA-binding protein (Imp) is a factor involved in the male-specific splicing of Bmdsx. In this study, we found that the male-specific Imp mRNA is formed as a result of the inclusion of exon 8 and the promoter-distal poly(A) site choice, whereas non-sex-specific polyadenylation occurs at the promoter-proximal poly(A) site downstream of exon 7. Recent studies revealed that Drosophila Sxl, tra in several dipteran and hymenopteran insects, and fem in Apis mellifera, play a central role in sex determination and maintain their productive mode of expression via an autoregulatory function. To determine whether Imp protein is required for the maintenance of the male-specific splicing of its own pre-mRNA, we knocked down endogenous Imp in male cells and assessed the male-specific splicing of an exogenous Imp minigene. Knockdown of endogenous Imp inhibited the male-specific splicing of the Imp minigene transcript. In contrast, overexpression of Imp in female cells induced the male-specific splicing of the Imp minigene transcript. Moreover, deletion of adenine-rich (A-rich) sequences located downstream of the proximal poly(A) site repressed the male-specific splicing of the Imp minigene transcript. Finally, gel shift analysis demonstrated that Imp binds to the A-rich sequences. These data suggest that Imp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA.

  10. The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

    PubMed

    Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

    2017-01-29

    Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.

  11. The human splicing code reveals new insights into the genetic determinants of disease

    PubMed Central

    Xiong, Hui Y.; Alipanahi, Babak; Lee, Leo J.; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K.C.; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S.; Hughes, Timothy R.; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R.; Jojic, Nebojsa; Scherer, Stephen W.; Blencowe, Benjamin J.; Frey, Brendan J.

    2015-01-01

    Introduction Advancing whole-genome precision medicine requires understanding how gene expression is altered by genetic variants, especially those that are outside of protein-coding regions. We developed a computational technique that scores how strongly genetic variants alter RNA splicing, a critical step in gene expression whose disruption contributes to many diseases, including cancers and neurological disorders. A genome-wide analysis reveals tens of thousands of variants that alter splicing and are enriched with a wide range of known diseases. Our results provide insight into the genetic basis of spinal muscular atrophy, hereditary nonpolyposis colorectal cancer and autism spectrum disorder. Methods We used machine learning to derive a computational model that takes as input DNA sequences and applies general rules to predict splicing in human tissues. Given a test variant, our model computes a score that predicts how much the variant disrupts splicing. The model was derived in such a way that it can be used to study diverse diseases and disorders, and to determine the consequences of common, rare, and even spontaneous variants. Results Our technique is able to accurately classify disease-causing variants and provides insights into the role of aberrant splicing in disease. We scored over 650,000 DNA variants and found that disease-causing variants have higher scores than common variants and even those associated with disease in genome-wide association studies. Our model predicts substantial and unexpected aberrant splicing due to variants within introns and exons, including those far from the splice site. For example, among intronic variants that are more than 30 nucleotides away from a splice site, known disease variants alter splicing nine times more often than common variants; among missense exonic disease variants, those that least impact protein function are over five times more likely to alter splicing than other variants. Autism has been associated with

  12. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  13. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  14. Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

    SciTech Connect

    Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki; Yagi, Hiroaki; Shigaki-Miyamoto, Kaya; Toyota, Syukichi; Azuma, Yuko; Igarashi, Masayuki; Tani, Tokio

    2014-03-28

    Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

  15. Characterization of a splicing mutation in group A xeroderma pigmentosum

    SciTech Connect

    Satokata, Ichiro; Tanaka, Kiyoji; Miura, Naoyuki; Miyamoto, Iwai; Okada, Yoshio ); Satoh, Yoshiaki Tokyo Medical and Dental Univ. ); Kondo, Seiji )

    1990-12-01

    The molecular basis of group A xeroderma pigmentosum (WP) was investigated by comparison of the nucleotide sequences of multiple clones of the XP group A complementing gene (XPAC) from a patient with group A XP with that of a normal gene. The clones showed a G {r arrow} C substitution at the 3{prime} splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotide to AC. Nucleotide sequencing of cDNAs amplified by the polymerase chain reaction revealed that this single base substitution abolishes the canonical 3{prime} splice site, thus creating two abnormally spliced mRNA forms. The larger form is identical with normal mRNA except for a dinucleotide deletion at the 5{prime} end of exon 4. This deletion results in a frameshift with premature translation termination in exon 4. The smaller form has a deletion of the entire exon 3 and the dinucleotide at the 5{prime} end of exon 4. The result of a transfection study provided additional evidence that this single base substitution is the disease-causing mutation. This single base substitution creates a new cleavage site for the restriction nuclease AlwNI. Analysis of AlwNI restriction fragment length polymorphism showed a high frequency of this mutation in Japanese patients with group A XP: 16 of 21 unrelated Japanese patients were homozygous and 4 were heterozygous for this mutation. However, 11 Caucasians and 2 Blacks with group A XP did not have this mutant allele. The polymorphic AlwNI restriction fragments are concluded to be useful for diagnosis of group A XP in Japanese subjects, including prenatal cases and carriers.

  16. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

    PubMed

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  17. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles

    NASA Astrophysics Data System (ADS)

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  18. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.

  19. Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

    PubMed

    Kjellman, Christian; Honeth, Gabriella; Järnum, Sofia; Lindvall, Magnus; Darabi, Anna; Nilsson, Ingar; Edvardsen, Klaus; Salford, Leif G; Widegren, Bengt

    2004-03-03

    Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

  20. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

    PubMed Central

    Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

  1. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  2. Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants

    PubMed Central

    Shang, Xudong; Cao, Ying; Ma, Ligeng

    2017-01-01

    Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism’s transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants. PMID:28230724

  3. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

    PubMed

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A; Khoder, Mamdouh I; Shamy, Magdy; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-07-01

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing.

  4. A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells

    PubMed Central

    Ajiro, Masahiko; Jia, Rong; Yang, Yanqin; Zhu, Jun; Zheng, Zhi-Ming

    2016-01-01

    Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis. PMID:26704980

  5. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  6. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.

  7. Cryptic splice sites and split genes.

    PubMed

    Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M; Tatusova, Tatiana; Dibb, Nick J

    2011-08-01

    We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes.

  8. Cryptic splice sites and split genes

    PubMed Central

    Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M.; Tatusova, Tatiana; Dibb, Nick J.

    2011-01-01

    We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes. PMID:21470962

  9. Pharmacology of Modulators of Alternative Splicing

    PubMed Central

    Morris, Jonathan C.; Oltean, Sebastian; Donaldson, Lucy F.

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. PMID:28034912

  10. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  11. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities

    PubMed Central

    Zhang, Jian; Lieu, Yen K.; Ali, Abdullah M.; Penson, Alex; Reggio, Kathryn S.; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L.

    2015-01-01

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein–protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting. PMID:26261309

  12. Insulin-inducible changes in the relative ratio of PTP1B splice variants.

    PubMed

    Sell, S M; Reese, D

    1999-03-01

    The skeletal muscle activity of protein tyrosine phosphates 1B (PTP1B), a modulator of insulin and IGF-1 signaling, is reduced in obese nondiabetic subjects and in subjects with type 2 diabetes in comparison with leaner, nondiabetic controls. PTP1B mRNA, like many other signaling molecules, including the insulin receptor, is alternatively spliced. Since we have shown that the ratio of the insulin receptor splice variants is modulated by insulin in vitro and is related to insulin levels in vivo, we hypothesized that the relative ratios of the alternatively spliced PTP1B mRNA might also vary in humans in proportion to the degree of hyperinsulinemia. This was tested in 21 nondiabetic Pima Indians, a population at increased risk for obesity and type 2 diabetes. The relative ratio of the PTP1B splice variants was quantified using RT-PCR of total RNA extracted from fractionated monocytes. The ratio of the splice variants was positively correlated with fasting plasma insulin concentration (r = 0.757; P = 0.0001), 2-h plasma insulin concentration following an oral glucose tolerance test (r = 0.614; P = 0.01, n = 16), and percentage of body fat (r = 0.746; P = 0.0001). These data indicate that variability in the ratio of the two splice variants is due, in part, to in vivo levels of chronic hyperinsulinemia. This simple, noninvasive assay is therefore a potential biomarker for chronic hyperinsulinemia, similar to the HbAlc assay in use to monitor glucose management in diabetic patients.

  13. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    PubMed

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  14. Splicing-directed therapy in a new mouse model of human accelerated aging.

    PubMed

    Osorio, Fernando G; Navarro, Claire L; Cadiñanos, Juan; López-Mejía, Isabel C; Quirós, Pedro M; Bartoli, Catherine; Rivera, José; Tazi, Jamal; Guzmán, Gabriela; Varela, Ignacio; Depetris, Danielle; de Carlos, Félix; Cobo, Juan; Andrés, Vicente; De Sandre-Giovannoli, Annachiara; Freije, José M P; Lévy, Nicolas; López-Otín, Carlos

    2011-10-26

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino-based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide-based therapies for treating human diseases of accelerated aging.

  15. LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development[W

    PubMed Central

    Perea-Resa, Carlos; Hernández-Verdeja, Tamara; López-Cobollo, Rosa; Castellano, María del Mar; Salinas, Julio

    2012-01-01

    In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay. PMID:23221597

  16. The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

    PubMed

    Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

    2015-05-01

    When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

  17. Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

    PubMed Central

    Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human

  18. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    SciTech Connect

    Iwasaki, Ryohei; Kiuchi, Hiroki; Ihara, Masaki; Mori, Toshihiro; Kawakami, Masayuki; Ueda, Hiroshi

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  19. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  20. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    PubMed Central

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

  1. Structural requirements for selection of 5'- and 3' splice sites of group II introns.

    PubMed Central

    Wallasch, C; Mörl, M; Niemer, I; Schmelzer, C

    1991-01-01

    The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro. It could recently be shown that self-splicing of this intron is fully reversible in vitro. In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA. The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1). We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification. Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e. insertion is one nt downstream or upstream of the 3' end of IBS1. This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1. To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions. Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and, ii) the first nucleotide(s) of the 3' exon play an important role in defining the 3' splice site. Images PMID:2062646

  2. RNA splicing manipulation: strategies to modify gene expression for a variety of therapeutic outcomes.

    PubMed

    Wilton, Steve D; Fletcher, Susan

    2011-08-01

    Antisense oligomers initially showed promise as compounds to modify gene expression, primarily through RNaseH induced degradation of the target transcript. Expansion of the field has led to new chemistries capable of invoking different mechanisms, including suppression of protein synthesis by translational blockade and gene silencing using short interfering RNAs. It is now apparent that the majority of the eukaryotic genome is transcribed and non-protein coding RNAs have been implicated in the regulation of gene expression at many levels. This review considers potential therapeutic applications of antisense oligomers to modify gene expression, primarily by interfering with the process of exon recognition and intron removal during gene transcript splicing. While suppression of gene expression will be necessary to address some conditions, it is likely that antisense oligomer splice modification will have extensive clinical application. Pre-mRNA splicing is a tightly co-ordinated, multifactorial process that can be disrupted by antisense oligomers in a highly specific manner to suppress aberrant splicing, remove exons to by-pass nonsense or frame-shifting mutations or influence exon selection to alter spliceoform ratios. Manipulation of splicing patterns has been applied to a diverse range of conditions, including b-thalassemia, Duchenne muscular dystrophy, spinal muscular atrophy and certain cancers. Alternative exon usage has been identified as a major mechanism for generating diversity from a limited repertoire of genes in higher eukaryotes. Considering that the majority of all human primary gene transcripts are reportedly alternatively spliced, intervention at the level of pre-mRNA processing is likely to become increasingly significant in the fight against genetic and acquired disorders.

  3. How To Measure Gravitational Aberration?

    NASA Astrophysics Data System (ADS)

    Krizek, M.; Solcova, A.

    2007-08-01

    In 1905, Henri Poincaré predicted the existence of gravitational waves and assumed that their speed c[g] would be that of the speed of light c. If the gravitational aberration would also have the same magnitude as the aberration of light, we would observe several paradoxical phenomena. For instance, the orbit of two bodies of equal mass would be unstable, since two attractive forces arise that are not in line and hence form a couple. This tends to increase the angular momentum, period, and total energy of the system. This can be modelled by a system of ordinary differential equations with delay. A big advantage of computer simulation is that we can easily perform many test for various possible values of the speed of gravity [1]. In [2], Carlip showed that gravitational aberration in general relativity is almost cancelled out by velocity-dependent interactions. This means that rays of sunlight are not parallel to the attractive gravitational force of the Sun, i.e., we do not see the Sun in the direction of its attractive force, but slightly shifted about an angle less than 20``. We show how the actual value of the gravitational aberration can be obtained by measurement of a single angle at a suitable time instant T corresponding to the perihelion of an elliptic orbit. We also derive an a priori error estimate that expresses how acurately T has to be determined to attain the gravitational aberration to a prescribed tolerance. [1] M. Křížek: Numerical experience with the finite speed of gravitational interaction, Math. Comput. Simulation 50 (1999), 237-245. [2] S. Carlip: Aberration and the speed of gravity, Phys. Lett. A 267 (2000), 81-87.

  4. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

  5. Epilepsy caused by an abnormal alternative splicing with dosage effect of the SV2A gene in a chicken model.

    PubMed

    Douaud, Marine; Feve, Katia; Pituello, Fabienne; Gourichon, David; Boitard, Simon; Leguern, Eric; Coquerelle, Gérard; Vieaud, Agathe; Batini, Cesira; Naquet, Robert; Vignal, Alain; Tixier-Boichard, Michèle; Pitel, Frédérique

    2011-01-01

    Photosensitive reflex epilepsy is caused by the combination of an individual's enhanced sensitivity with relevant light stimuli, such as stroboscopic lights or video games. This is the most common reflex epilepsy in humans; it is characterized by the photoparoxysmal response, which is an abnormal electroencephalographic reaction, and seizures triggered by intermittent light stimulation. Here, by using genetic mapping, sequencing and functional analyses, we report that a mutation in the acceptor site of the second intron of SV2A (the gene encoding synaptic vesicle glycoprotein 2A) is causing photosensitive reflex epilepsy in a unique vertebrate model, the Fepi chicken strain, a spontaneous model where the neurological disorder is inherited as an autosomal recessive mutation. This mutation causes an aberrant splicing event and significantly reduces the level of SV2A mRNA in homozygous carriers. Levetiracetam, a second generation antiepileptic drug, is known to bind SV2A, and SV2A knock-out mice develop seizures soon after birth and usually die within three weeks. The Fepi chicken survives to adulthood and responds to levetiracetam, suggesting that the low-level expression of SV2A in these animals is sufficient to allow survival, but does not protect against seizures. Thus, the Fepi chicken model shows that the role of the SV2A pathway in the brain is conserved between birds and mammals, in spite of a large phylogenetic distance. The Fepi model appears particularly useful for further studies of physiopathology of reflex epilepsy, in comparison with induced models of epilepsy in rodents. Consequently, SV2A is a very attractive candidate gene for analysis in the context of both mono- and polygenic generalized epilepsies in humans.

  6. Pokeweed antiviral protein alters splicing of HIV-1 RNAs, resulting in reduced virus production.

    PubMed

    Zhabokritsky, Alice; Mansouri, Sheila; Hudak, Katalin A

    2014-08-01

    Processing of HIV-1 transcripts results in three populations in the cytoplasm of infected cells: full-length RNA, singly spliced, and multiply spliced RNAs. Rev, regulator of virion expression, is an essential regulatory protein of HIV-1 required for transporting unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm. Export allows these RNAs to be translated and the full-length RNA to be packaged into virus particles. In our study, we investigate the activity of pokeweed antiviral protein (PAP), a glycosidase isolated from the pokeweed plant Phytolacca americana, on the processing of viral RNAs. We show that coexpression of PAP with a proviral clone alters the splicing ratio of HIV-1 RNAs. Specifically, PAP causes the accumulation of multiply spliced 2-kb RNAs at the expense of full-length 9-kb and singly spliced 4-kb RNAs. The change in splicing ratio is due to a decrease in activity of Rev. We show that PAP depurinates the rev open reading frame and that this damage to the viral RNA inhibits its translation. By decreasing Rev expression, PAP indirectly reduces the availability of full-length 9-kb RNA for packaging and translation of the encoded structural proteins required for synthesis of viral particles. The decline we observe in virus protein expression is not due to cellular toxicity as PAP did not diminish translation rate. Our results describing the reduced activity of a regulatory protein of HIV-1, with resulting change in virus mRNA ratios, provides new insight into the antiviral mechanism of PAP.

  7. Splicing repression allows the gradual emergence of new Alu-exons in primate evolution

    PubMed Central

    Attig, Jan; Ruiz de los Mozos, Igor; Haberman, Nejc; Wang, Zhen; Emmett, Warren; Zarnack, Kathi; König, Julian; Ule, Jernej

    2016-01-01

    Alu elements are retrotransposons that frequently form new exons during primate evolution. Here, we assess the interplay of splicing repression by hnRNPC and nonsense-mediated mRNA decay (NMD) in the quality control and evolution of new Alu-exons. We identify 3100 new Alu-exons and show that NMD more efficiently recognises transcripts with Alu-exons compared to other exons with premature termination codons. However, some Alu-exons escape NMD, especially when an adjacent intron is retained, highlighting the importance of concerted repression by splicing and NMD. We show that evolutionary progression of 3' splice sites is coupled with longer repressive uridine tracts. Once the 3' splice site at ancient Alu-exons reaches a stable phase, splicing repression by hnRNPC decreases, but the exons generally remain sensitive to NMD. We conclude that repressive motifs are strongest next to cryptic exons and that gradual weakening of these motifs contributes to the evolutionary emergence of new alternative exons. DOI: http://dx.doi.org/10.7554/eLife.19545.001 PMID:27861119

  8. SRRM4-dependent neuron-specific alternative splicing of protrudin transcripts regulates neurite outgrowth

    PubMed Central

    Ohnishi, Takafumi; Shirane, Michiko; Nakayama, Keiichi I.

    2017-01-01

    Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts. PMID:28106138

  9. Global impact of RNA splicing on transcriptome remodeling in the heart.

    PubMed

    Gao, Chen; Wang, Yibin

    2012-08-01

    In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

  10. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  11. RNA Splicing in a New Rhabdovirus from Culex Mosquitoes▿†

    PubMed Central

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-01-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae. PMID:21507977

  12. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing

    PubMed Central

    Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G.; Mammi, Pablo; Yanovsky, Marcelo J.; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V.

    2016-01-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. PMID:27575636

  13. Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands

    PubMed Central

    Zubović, Lorena; Baralle, Marco; Baralle, Francisco E.

    2012-01-01

    Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Nav 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1. PMID:22434879

  14. Functional Integration of mRNA Translational Control Programs

    PubMed Central

    MacNicol, Melanie C.; Cragle, Chad E.; Arumugam, Karthik; Fosso, Bruno; Pesole, Graziano; MacNicol, Angus M.

    2015-01-01

    Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease. PMID:26197342

  15. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy

    PubMed Central

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients’ subgroup, are explored. PMID:28164087

  16. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy.

    PubMed

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo; Bria, Emilio

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients' subgroup, are explored.

  17. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html.

  18. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events

    PubMed Central

    Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M.; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N.; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D.; Gazdar, Adi F.; Stern, Howard M.; Hoeflich, Klaus P.; Wu, Thomas D.; Settleman, Jeff; de Sauvage, Frederic J.; Gentleman, Robert C.; Neve, Richard M.; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S.; Zhang, Zemin

    2012-01-01

    Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberra