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Sample records for alter rna duplex

  1. Probing the transcription mechanisms of reovirus cores with molecules that alter RNA duplex stability.

    PubMed

    Demidenko, Alexander A; Nibert, Max L

    2009-06-01

    The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps. PMID:19297468

  2. Antisense RNA: Function and Fate of Duplex RNA in Cells of Higher Eukaryotes

    PubMed Central

    Kumar, Madhur; Carmichael, Gordon G.

    1998-01-01

    There is ample evidence that cells of higher eukaryotes express double-stranded RNA molecules (dsRNAs) either naturally or as the result of viral infection or aberrant, bidirectional transcriptional readthrough. These duplex molecules can exist in either the cytoplasmic or nuclear compartments. Cells have evolved distinct ways of responding to dsRNAs, depending on the nature and location of the duplexes. Since dsRNA molecules are not thought to exist naturally within the cytoplasm, dsRNA in this compartment is most often associated with viral infections. Cells have evolved defensive strategies against such molecules, primarily involving the interferon response pathway. Nuclear dsRNA, however, does not induce interferons and may play an important posttranscriptional regulatory role. Nuclear dsRNA appears to be the substrate for enzymes which deaminate adenosine residues to inosine residues within the polynucleotide structure, resulting in partial or full unwinding. Extensively modified RNAs are either rapidly degraded or retained within the nucleus, whereas transcripts with few modifications may be transported to the cytoplasm, where they serve to produce altered proteins. This review summarizes our current knowledge about the function and fate of dsRNA in cells of higher eukaryotes and its potential manipulation as a research and therapeutic tool. PMID:9841677

  3. ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis

    PubMed Central

    Zhang, Xiaoming; Niu, DongDong; Carbonell, Alberto; Wang, Airong; Lee, Angel; Tun, Vinnary; Wang, Zonghua; Carrington, James C.; Chang, Chia-en A.; Jin, Hailing

    2014-01-01

    Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5′-terminal nucleotide is important for sRNA sorting into different AGOs. Here, we show that miRNA duplex structure also contributes to miRNA sorting. Base-pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favors miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modeling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base-pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2. PMID:25406978

  4. All-atom crystal simulations of DNA and RNA duplexes

    PubMed Central

    Liu, Chunmei; Janowski, Pawel A.; Case, David A.

    2014-01-01

    Background Molecular dynamics simulations can complement experimental measures of structure and dynamics of biomolecules. The quality of such simulations can be tested by comparisons to models refined against experimental crystallographic data. Methods We report simulations of a DNA and RNA duplex in their crystalline environment. The calculations mimic the conditions for PDB entries 1D23 [d(CGATCGATCG)2] and 1RNA [(UUAUAUAUAUAUAA)2], and contain 8 unit cells, each with 4 copies of the Watson-Crick duplex; this yields in aggregate 64 µs of duplex sampling for DNA and 16 µs for RNA. Results The duplex structures conform much more closely to the average structure seen in the crystal than do structures extracted from a solution simulation with the same force field. Sequence-dependent variations in helical parameters, and in groove widths, are largely maintained in the crystal structure, but are smoothed out in solution. However, the integrity of the crystal lattice is slowly degraded in both simulations, with the result that the interfaces between chains become heterogeneous. This problem is more severe for the DNA crystal, which has fewer inter-chain hydrogen bond contacts than does the RNA crystal. Conclusions Crystal simulations using current force fields reproduce many features of observed crystal structures, but suffer from a gradual degradation of the integrity of the crystal lattice. General significance The results offer insights into force-field simulations that tests their ability to preserve weak interactions between chains, which will be of importance also in non-crystalline applications that involve binding and recognition. PMID:25255706

  5. Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

    PubMed Central

    Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

    2007-01-01

    Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription. PMID:17356699

  6. mRNA-mRNA duplexes that autoelicit Staufen1-mediated mRNA decay.

    PubMed

    Gong, Chenguang; Tang, Yalan; Maquat, Lynne E

    2013-10-01

    We report a new mechanism by which human mRNAs cross-talk: an Alu element in the 3' untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA, thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3'-UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3'-UTR SBS created by mRNA-mRNA base-pairing, we show that SMD targets both mRNAs in the duplex, provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the functional importance of mRNA-mRNA-triggered SMD in cell migration and invasion. PMID:24056942

  7. Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

    PubMed Central

    Szabat, Marta; Pedzinski, Tomasz; Czapik, Tomasz; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes. PMID:26579720

  8. Helically Assembled Pyrene Arrays on an RNA Duplex That Exhibit Circularly Polarized Luminescence with Excimer Formation.

    PubMed

    Nakamura, Mitsunobu; Suzuki, Junpei; Ota, Fuyuki; Takada, Tadao; Akagi, Kazuo; Yamana, Kazushige

    2016-06-27

    Circularly polarized luminescence (CPL) was observed in pyrene zipper arrays helically arranged on an RNA duplex. Hybridization of complementary RNA strands having multiple (two to five) 2'-O-pyrenylmethyl modified nucleosides affords an RNA duplex with normal thermal stability. The pyrene fluorophores are assembled like a zipper in a well-defined helical manner along the axis of RNA duplex, which, upon 350 nm UV illumination, resulted in CPL emission with pyrene excimer formation. CPL (glum ) levels observed for the pyrene arrays in dilute aqueous solution were +2×10(-2) -+3.5×10(-2) , which are comparable with |glum | for chiral organic molecules and related systems. The positive CPL signals are consistent with a right-handed helical structure. Temperature dependence on CPL emission indicates that the stable rigid RNA structure is responsible for the strong CPL signals. The single pyrene-modified RNA duplex did not show any CPL signal. PMID:27150679

  9. Structure of an A-form RNA duplex obtained by degradation of 6S RNA in a crystallization droplet

    PubMed Central

    Kondo, Jiro; Dock-Bregeon, Anne-Catherine; Willkomm, Dagmar K.; Hartmann, Roland K.; Westhof, Eric

    2013-01-01

    In the course of a crystallographic study of a 132 nt variant of Aquifex aeolicus 6S RNA, a crystal structure of an A-form RNA duplex containing 12 base pairs was solved at a resolution of 2.6 Å. In fact, the RNA duplex is part of the 6S RNA and was obtained by accidental but precise degradation of the 6S RNA in a crystallization droplet. 6S RNA degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. The RNA oligomers obtained form regular A-form duplexes containing three GoU wobble-type base pairs, one of which engages in intermolecular contacts through a ribose-zipper motif at the crystal-packing interface. PMID:23722840

  10. Structure of an A-form RNA duplex obtained by degradation of 6S RNA in a crystallization droplet.

    PubMed

    Kondo, Jiro; Dock-Bregeon, Anne Catherine; Willkomm, Dagmar K; Hartmann, Roland K; Westhof, Eric

    2013-06-01

    In the course of a crystallographic study of a 132 nt variant of Aquifex aeolicus 6S RNA, a crystal structure of an A-form RNA duplex containing 12 base pairs was solved at a resolution of 2.6 Å. In fact, the RNA duplex is part of the 6S RNA and was obtained by accidental but precise degradation of the 6S RNA in a crystallization droplet. 6S RNA degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. The RNA oligomers obtained form regular A-form duplexes containing three GoU wobble-type base pairs, one of which engages in intermolecular contacts through a ribose-zipper motif at the crystal-packing interface. PMID:23722840

  11. Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

    PubMed Central

    Zhou, Yuan; Kierzek, Elzbieta; Loo, Zi Ping; Antonio, Meraldo; Yau, Yin Hoe; Chuah, York Wieo; Geifman-Shochat, Susana; Kierzek, Ryszard; Chen, Gang

    2013-01-01

    Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2′-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine–pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA. PMID:23658228

  12. Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides.

    PubMed

    Zhou, Yuan; Kierzek, Elzbieta; Loo, Zi Ping; Antonio, Meraldo; Yau, Yin Hoe; Chuah, York Wieo; Geifman-Shochat, Susana; Kierzek, Ryszard; Chen, Gang

    2013-07-01

    Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2'-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine-purine-pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2'-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA. PMID:23658228

  13. Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

    PubMed Central

    Schneck, P K; Staudenbauer, W L; Hofschneider, P H

    1976-01-01

    Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

  14. The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

    PubMed Central

    Eamens, Andrew L.; Smith, Neil A.; Curtin, Shaun J.; Wang, Ming-Bo; Waterhouse, Peter M.

    2009-01-01

    In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 5′ thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms. PMID:19861421

  15. Duplex End Breathing Determines Serum Stability and Intracellular Potency of siRNA-Au NPs

    PubMed Central

    Patel, Pinal C.; Hao, Liangliang; Yeung, Weng Si Au; Mirkin, Chad A.

    2011-01-01

    Structural requirements of siRNA-functionalized gold nanoparticles (siRNA-Au NPs) for Dicer recognition and serum stability were studied. We show that the 3′ overhang on the nucleic acids of these particles is preferentially recognized by Dicer but also makes the siRNA duplexes more susceptible to non-specific serum degradation. Dicer and serum nucleases show lower preference for blunt duplexes as opposed to those with 3′ overhangs. Importantly, gold nanoparticles functionalized with blunt duplexes with relatively less thermal breathing are up to 15 times more stable against serum degradation without compromising Dicer recognition. This increased stability leads to a 300% increase in cellular uptake of siRNA-Au NPs and improved gene knockdown. PMID:21630673

  16. RNA chaperones stimulate formation and yield of the U3 snoRNA-pre-rRNA duplexes needed for eukaryotic ribosome biogenesis

    PubMed Central

    Gérczei, Tímea; Shah, Binal N.; Manzo, Anthony J.; Walter, Nils G.; Correll, Carl C.

    2010-01-01

    To satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells, short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor ribosomal RNA (pre-rRNA) must form quickly and with high yield. These interactions, designated the U3-ETS and U3-18S duplexes, are essential to initiate the processing of small subunit rRNA. Previously, we showed in vitro that duplexes corresponding to those in Saccharomyces cerevisiae are only observed after addition of one of two proteins: Imp3p or Imp4p. Here, we used fluorescence-based and other in vitro assays to determine whether these proteins possess RNA chaperone activities and to assess whether these activities are sufficient to satisfy the duplex yield and rate requirements expected in vivo. Assembly of both proteins with the U3 snoRNA into a chaperone complex destabilizes a U3-stem structure, apparently to expose its 18S base-pairing site. As a result, the chaperone complex accelerates formation of the U3-18S duplex from an undetectable rate to one comparable to the intrinsic rate observed for hybridizing short duplexes. The chaperone complex also stabilizes the U3-ETS duplex by 2.7 kcal/mol. These chaperone activities provide high U3-ETS duplex yield and rapid U3-18S duplex formation over a broad concentration range to help ensure that the U3-pre-rRNA interactions limit neither ribosome biogenesis nor rapid cell growth. The thermodynamic and kinetic framework used is general and thus suitable to investigate the mechanism of action of other RNA chaperones. PMID:19482034

  17. Nearest-neighbor parameters for 7-deaza-adenosine·uridine base pairs in RNA duplexes.

    PubMed

    Richardson, Katherine E; Znosko, Brent M

    2016-06-01

    One of the major limitations in RNA structure prediction is the lack of information about the effect of nonstandard nucleotides on stability. The nonstandard nucleotide 7-deaza-adenosine (7DA) is a naturally occurring analog of adenosine that has been studied for medicinal purposes and is commonly referred to as tubercidin. In 7DA, the nitrogen in the 7 position of adenosine is replaced by a carbon. Differences in RNA duplex stability due to the removal of this nitrogen can be attributed to a possible change in hydration and a difference in base stacking interactions resulting from changes in the electrostatics of the ring. In order to determine how 7DA affects the stability of RNA, optical melting experiments were conducted on RNA duplexes that contain either internal or terminal 7DA·U pairs with all possible nearest-neighbor combinations. On average, duplexes containing 7DA·U pairs are 0.43 and 0.07 kcal/mol less stable than what is predicted for the same duplex containing internal and terminal A-U pairs, respectively. Thermodynamic parameters for all nearest-neighbor combinations of 7DA·U pairs were derived from the data. These parameters can be used to more accurately predict the secondary structure and stability of RNA duplexes containing 7DA·U pairs. PMID:27099368

  18. Direct Duplex Detection: An Emerging Tool in the RNA Structure Analysis Toolbox.

    PubMed

    Weidmann, Chase A; Mustoe, Anthony M; Weeks, Kevin M

    2016-09-01

    While a variety of powerful tools exists for analyzing RNA structure, identifying long-range and intermolecular base-pairing interactions has remained challenging. Recently, three groups introduced a high-throughput strategy that uses psoralen-mediated crosslinking to directly identify RNA-RNA duplexes in cells. Initial application of these methods highlights the preponderance of long-range structures within and between RNA molecules and their widespread structural dynamics. PMID:27427309

  19. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    PubMed Central

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  20. Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine.

    PubMed Central

    Martinis, S A; Schimmel, P

    1992-01-01

    RNA hairpin helices whose sequences are based on the acceptor stems of alanine and histidine tRNAs are specifically aminoacylated with their cognate amino acids. In these examples, major determinants for the identities of the respective tRNAs reside in the acceptor stem; the anticodon and other parts of the tRNA are dispensable for aminoacylation. In contrast, the anticodon is a major determinant for the identity of a methionine tRNA. RNA hairpin helices and hybrid duplexes that reconstruct the acceptor-T psi C stem and the acceptor stem, respectively, of methionine tRNA were investigated here for aminoacylation with methionine. Direct visualization of the aminoacylated RNA product on an acidic polyacrylamide gel by phosphor imaging demonstrated specific aminoacylation with substrates that contained as few as 7 base pairs. No aminoacylation with methionine was detected with several analogous RNA substrates whose sequences were based on noncognate tRNAs. While the efficiency of aminoacylation is reduced by orders of magnitude relative to methionine tRNA, the results establish that specific aminoacylation with methionine of small duplex substrates can be achieved without the anticodon or other domains of the tRNA. The results, combined with earlier studies, suggest a highly specific adaptation of the structures of aminoacyl-tRNA synthetases to the acceptor stems of their cognate tRNAs, resulting in a relationship between the nucleotide sequences/structures of small RNA duplexes and specific amino acids. Images PMID:1729719

  1. Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

    PubMed Central

    Kibler-Herzog, L; Zon, G; Uznanski, B; Whittier, G; Wilson, W D

    1991-01-01

    The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations. PMID:1711677

  2. Base pairing and structural insights into the 5-formylcytosine in RNA duplex

    PubMed Central

    Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O.; Chen, Doris; Sheng, Jia

    2016-01-01

    5-Formylcytidine (f5C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m5C) through 5-hydroxymethylcytidine (hm5C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f5C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5′-GUA(f5C)GUAC-3′]2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f5C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

  3. Base pairing and structural insights into the 5-formylcytosine in RNA duplex.

    PubMed

    Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O; Chen, Doris; Sheng, Jia

    2016-06-01

    5-Formylcytidine (f(5)C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m(5)C) through 5-hydroxymethylcytidine (hm(5)C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f(5)C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5'-GUA(f(5)C)GUAC-3']2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f(5)C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

  4. Newcastle Disease Virus-Specific RNA: Polyacrylamide Gel Analysis of Single-Stranded RNA and Hybrid Duplexes

    PubMed Central

    Kaverin, Nicolai V.; Varich, Natalia L.

    1974-01-01

    Newcastle disease virus-specific [3H]uridine-labeled 18S RNA was resolved by polyacrylamide gel electrophoresis into several components with molecular weights from 450,000 to 840,000. The analysis of 35 and 24S virus-specific RNA also revealed several components in each sedimentational class. The conversion of 18S RNA into double-stranded form by hybridization with an excess of unlabeled virion RNA improved the resolution in polyacrylamide gels and revealed at least six distinct components. The same six classes of hybrid duplexes were revealed when 32P-labeled 50S virion RNA was hybridized with an excess of 18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexes to the analysis of viral genome structure is discussed. PMID:4855736

  5. Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines.

    PubMed Central

    Minasov, G; Matulic-Adamic, J; Wilds, C J; Haeberli, P; Usman, N; Beigelman, L; Egli, M

    2000-01-01

    Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs. PMID:11105752

  6. Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

    PubMed

    Seio, Kohji; Tokugawa, Munefumi; Tsunoda, Hirosuke; Ohkubo, Akihiro; Arisaka, Fumio; Sekine, Mitsuo

    2013-12-15

    The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration. PMID:24183539

  7. Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding.

    PubMed

    Ennifar, Eric; Bernacchi, Serena; Wolff, Philippe; Dumas, Philippe

    2007-09-01

    Halogenation of bases is a widespread method used for solving crystal structures of nucleic acids. However, this modification may have important consequences on RNA folding and thus on the success of crystallization. We have used a combination of UV thermal melting, steady-state fluorescence, X-ray crystallography, and gel electrophoresis techniques to study the influence of uridine halogenation (bromination or iodination) on the RNA folding. The HIV-1 Dimerization Initiation Site is an RNA hairpin that can adopt an alternative duplex conformation and was used as a model. We have shown that, unexpectedly, the RNA hairpin/duplex ratio is strongly dependent not only on the presence but also on the position of halogenation. PMID:17630326

  8. Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding

    PubMed Central

    Ennifar, Eric; Bernacchi, Serena; Wolff, Philippe; Dumas, Philippe

    2007-01-01

    Halogenation of bases is a widespread method used for solving crystal structures of nucleic acids. However, this modification may have important consequences on RNA folding and thus on the success of crystallization. We have used a combination of UV thermal melting, steady-state fluorescence, X-ray crystallography, and gel electrophoresis techniques to study the influence of uridine halogenation (bromination or iodination) on the RNA folding. The HIV-1 Dimerization Initiation Site is an RNA hairpin that can adopt an alternative duplex conformation and was used as a model. We have shown that, unexpectedly, the RNA hairpin/duplex ratio is strongly dependent not only on the presence but also on the position of halogenation. PMID:17630326

  9. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  10. Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

    PubMed Central

    Gaidamakov, Sergei A.; Gorshkova, Inna I.; Schuck, Peter; Steinbach, Peter J.; Yamada, Hirofumi; Crouch, Robert J.; Cerritelli, Susana M.

    2005-01-01

    Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA–DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA–DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication. PMID:15831789

  11. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.

    PubMed

    Lu, Zhipeng; Zhang, Qiangfeng Cliff; Lee, Byron; Flynn, Ryan A; Smith, Martin A; Robinson, James T; Davidovich, Chen; Gooding, Anne R; Goodrich, Karen J; Mattick, John S; Mesirov, Jill P; Cech, Thomas R; Chang, Howard Y

    2016-05-19

    RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT. PMID:27180905

  12. Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

    PubMed

    Barsky, D; Colvin, M E; Zon, G; Gryaznov, S M

    1997-02-15

    Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone. PMID:9016634

  13. Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

    PubMed Central

    Barsky, D; Colvin, M E; Zon, G; Gryaznov, S M

    1997-01-01

    Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone. PMID:9016634

  14. MiRduplexSVM: A High-Performing MiRNA-Duplex Prediction and Evaluation Methodology

    PubMed Central

    Karathanasis, Nestoras; Tsamardinos, Ioannis; Poirazi, Panayiota

    2015-01-01

    We address the problem of predicting the position of a miRNA duplex on a microRNA hairpin via the development and application of a novel SVM-based methodology. Our method combines a unique problem representation and an unbiased optimization protocol to learn from mirBase19.0 an accurate predictive model, termed MiRduplexSVM. This is the first model that provides precise information about all four ends of the miRNA duplex. We show that (a) our method outperforms four state-of-the-art tools, namely MaturePred, MiRPara, MatureBayes, MiRdup as well as a Simple Geometric Locator when applied on the same training datasets employed for each tool and evaluated on a common blind test set. (b) In all comparisons, MiRduplexSVM shows superior performance, achieving up to a 60% increase in prediction accuracy for mammalian hairpins and can generalize very well on plant hairpins, without any special optimization. (c) The tool has a number of important applications such as the ability to accurately predict the miRNA or the miRNA*, given the opposite strand of a duplex. Its performance on this task is superior to the 2nts overhang rule commonly used in computational studies and similar to that of a comparative genomic approach, without the need for prior knowledge or the complexity of performing multiple alignments. Finally, it is able to evaluate novel, potential miRNAs found either computationally or experimentally. In relation with recent confidence evaluation methods used in miRBase, MiRduplexSVM was successful in identifying high confidence potential miRNAs. PMID:25961860

  15. Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA.

    PubMed

    Zou, Tingting; Kizaki, Seiichiro; Pandian, Ganesh N; Sugiyama, Hiroshi

    2016-06-20

    To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B2 to duplex DNA could advance its design as an antitumor agent. PMID:27123891

  16. Synthesis of oligodiaminomannoses and analysis of their RNA duplex binding properties and their potential application as siRNA-based drugs.

    PubMed

    Iwata, Rintaro; Doi, Akiko; Maeda, Yusuke; Wada, Takeshi

    2015-09-28

    The synthesis of artificial cationic oligodiaminosaccharides, α-(1 → 4)-linked-2,6-diamino-2,6-dideoxy-d-mannopyranose oligomers (ODAMans), and their interactions with RNA duplexes are described. The monomer through the pentamer, all of which bear unnatural 2,6-diaminomannose moieties, were successfully prepared. UV melting and fluorescence anisotropy analyses revealed that the ODAMans bound and thermodynamically stabilized both 12mer RNA duplexes and an siRNA. Furthermore, it was clearly shown that the siRNA acquired substantial RNase A resistance due to its binding to the ODAMan 4mer. PMID:26256756

  17. Magnesium effect on premelting transitions in nucleic acids: DNA duplex and RNA hairpin models

    NASA Astrophysics Data System (ADS)

    Ottová, Pavla; Espinoza-Herrera, Shirly Josefina; Štěpánek, Josef

    2011-05-01

    The effect of magnesium ions on the conformational changes in the temperature region below the melting transition (premelting transitions) of a DNA duplex and an RNA hairpin was studied by difference Raman spectroscopy. The chosen model systems were the polynucleotide poly(dA)-poly(dT) duplex and the apical hairpin of the trans-activation response (TAR) element of HIV-1. Magnesium effect was displayed by differences of Raman spectra measured at various temperatures with and without magnesium ions. Our results have revealed that magnesium ions influence measurably the premelting transitions in both cases; the extent of the effect and its character is though quite different. In the case of the B' → B premelting transition of the polynucleotide poly(dA)-poly(dT) duplex, magnesium binds to the minor groove of the B but not of the B' form and acts again the rearrangement in the vicinity of the thymidine keto-groups. On the other hand, the magnesium effect on the TAR hairpin premelting consists in a weak support of the premelting structural change via more effective electrostatic shielding.

  18. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA.

    PubMed

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping

    2016-02-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction. PMID:26688865

  19. DUPLEX: A molecular mechanics program in torsion angle space for computing structures of DNA and RNA

    SciTech Connect

    Hingerty, B.E.

    1992-07-01

    DUPLEX produces energy minimized structures of DNA and RNA of any base sequence for single and double strands. The smallest subunits are deoxydinucleoside monophosphates, and up to 12 residues, single or double stranded can be treated. In addition, it can incorporate NMR derived interproton distances an constraints in the minimizations. Both upper and lower bounds for these distances can be specified. The program has been designed to run on a UNICOS Cray supercomputer, but should run, albeit slowly, on a laboratory computer such as a VAX or a workstation.

  20. Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

    PubMed Central

    Devi, Gitali; Yuan, Zhen; Lu, Yunpeng; Zhao, Yanli; Chen, Gang

    2014-01-01

    Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA2–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA2 triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA2–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA2–PNA triplex is significantly more stable than a DNA2–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications. PMID:24423869

  1. A crystallographic study of the binding of 13 metal ions to two related RNA duplexes

    PubMed Central

    Ennifar, Eric; Walter, Philippe; Dumas, Philippe

    2003-01-01

    Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K+, Pb2+, Mn2+, Ba2+, Ca2+, Cd2+, Sr2+, Zn2+, Co2+, Au3+ and Pt4+) and two hexammines [Co (NH3)6]3+ and [Ru (NH3)6]3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg2+, at ‘Hoogsteen’ sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH3)4]3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH3)6]3+ as a [Mg (H2O)6]2+ mimetic. Also very unexpected was the binding of the small Au3+ cation exactly between the Watson–Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium. PMID:12736317

  2. Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2′-O-methylation

    PubMed Central

    Appel, C. Denise; Maxwell, E. Stuart

    2007-01-01

    Archaeal box C/D sRNAs guide the 2′-O-methylation of target nucleotides using both terminal box C/D and internal C′/D′ RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson–Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson–Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg2+ concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D′ target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These observations suggested that unique structural features of the box C/D and C′/D′ RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation. PMID:17438123

  3. Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2'-O-methylation.

    PubMed

    Appel, C Denise; Maxwell, E Stuart

    2007-06-01

    Archaeal box C/D sRNAs guide the 2'-O-methylation of target nucleotides using both terminal box C/D and internal C'/D' RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson-Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg(2+) concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D' target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg(2+) concentrations. These observations suggested that unique structural features of the box C/D and C'/D' RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation. PMID:17438123

  4. Analysis of a ribonuclease H digestion of N3'-->P5' phosphoramidate-RNA duplexes by capillary gel electrophoresis.

    PubMed

    DeDionisio, L; Gryaznov, S M

    1995-07-01

    Phosphodiester oligonucleotides (ODNs) and their analogs are presently being investigated as potential antisense therapeutics in the treatment of viral infections and various forms of cancer. here, we would like to report results from an investigation of activity for a ribonuclease H (RNase H) mediated RNA digestion assay in the duplexes formed by an ODN or the ODN analog, N3'-->P5' phosphoramidate (3'-phosphoramidate), and complimentary RNA strands. Capillary gel electrophoresis (CGE) proved to be an effective method for determining RNA hydrolysis in the presence of RNase H. RNA and an ODN or RNA and a 3'-phosphoramidate were hybridized in a Tris-HCl, MgCl2 buffer at room temperature (RT) and incubated with RNase H. Digestions were carried out at RT or at 37 degrees C. Control samples were unhybridized RNA with RNase H, RNA without RNase H, and duplexes (RNA-ODN or 3'-phosphoramidate) without RNase H. All controls were incubated in Tris-HCl, MgCl2 buffer, and sample aliquots were analyzed at various time intervals. A homodecamer, (dT)10, was used as an internal standard to determine the relative migration time of the RNA strand. The final digestion products for the duplexes and the various controls were monitored by CGE. In addition, polyacrylamide gel electrophoresis (PAGE) was used in conjunction with Stains-All (staining) and a densitometric analysis to verify CGE results. PMID:7581876

  5. Comparing Charge Transport in Oligonucleotides: RNA:DNA Hybrids and DNA Duplexes.

    PubMed

    Li, Yuanhui; Artés, Juan M; Qi, Jianqing; Morelan, Ian A; Feldstein, Paul; Anantram, M P; Hihath, Joshua

    2016-05-19

    Understanding the electronic properties of oligonucleotide systems is important for applications in nanotechnology, biology, and sensing systems. Here the charge-transport properties of guanine-rich RNA:DNA hybrids are compared to double-stranded DNA (dsDNA) duplexes with identical sequences. The conductance of the RNA:DNA hybrids is ∼10 times higher than the equivalent dsDNA, and conformational differences are determined to be the primary reason for this difference. The conductance of the RNA:DNA hybrids is also found to decrease more rapidly than dsDNA when the length is increased. Ab initio electronic structure and Green's function-based density of states calculations demonstrate that these differences arise because the energy levels are more spatially distributed in the RNA:DNA hybrid but that the number of accessible hopping sites is smaller. These combination results indicate that a simple hopping model that treats each individual guanine as a hopping site is insufficient to explain both a higher conductance and β value for RNA:DNA hybrids, and larger delocalization lengths must be considered. PMID:27145167

  6. The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines.

    PubMed

    Kierzek, Elzbieta; Kierzek, Ryszard

    2003-08-01

    The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N6-alkyladenosines and 2-methylthio-N6-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A(Mod) base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3'-terminal unpaired nucleotide. Beside naturally modified adenosines such as N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A) and 2-methylthio-N6-methyladenosine (ms2m6A), we studied several artificial modifications to evaluate the steric and electronic effects of N6-alkyl substituents. Moreover, some N6-alkyladenosines and 2-methylthio-N6-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A(Mod) base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA. PMID:12888507

  7. RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

    PubMed Central

    Liu, Jing; Pendergraff, Hannah; Narayanannair, K. Jayaprakash; Lackey, Jeremy G.; Kuchimanchi, Satya; Rajeev, Kallanthottathil G.; Manoharan, Muthiah; Hu, Jiaxin; Corey, David R.

    2013-01-01

    Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing. PMID:23887934

  8. Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes

    NASA Astrophysics Data System (ADS)

    Kimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.

    2015-03-01

    Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10-3 to 10-5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

  9. Visualizing Transient Watson-Crick Like Mispairs in DNA and RNA Duplexes

    PubMed Central

    Kimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.

    2015-01-01

    Rare tautomeric and anionic nucleobases are believed to play fundamental biological roles but their prevalence and functional importance has remained elusive because they exist transiently, in low-abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10−3-10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases. PMID:25762137

  10. The X-ray Structures of Six Octameric RNA Duplexes in the Presence of Different Di- and Trivalent Cations

    PubMed Central

    Schaffer, Michelle F.; Peng, Guanya; Spingler, Bernhard; Schnabl, Joachim; Wang, Meitian; Olieric, Vincent; Sigel, Roland K. O.

    2016-01-01

    Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca2+, Mn2+, Co2+, Cu2+, Sr2+, and Tb3+. The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map. PMID:27355942

  11. The X-ray Structures of Six Octameric RNA Duplexes in the Presence of Different Di- and Trivalent Cations.

    PubMed

    Schaffer, Michelle F; Peng, Guanya; Spingler, Bernhard; Schnabl, Joachim; Wang, Meitian; Olieric, Vincent; Sigel, Roland K O

    2016-01-01

    Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca(2+), Mn(2+), Co(2+), Cu(2+), Sr(2+), and Tb(3+). The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map. PMID:27355942

  12. ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

    NASA Astrophysics Data System (ADS)

    Birrento, Monica L.; Bryan, Tracy M.; Samosorn, Siritron; Beck, Jennifer L.

    2015-07-01

    Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine ( 1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of ( 1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand ( 1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

  13. Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

    PubMed

    Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. PMID:23968861

  14. Structural characterization of a dimer of RNA duplexes composed of 8-bromoguanosine modified CGG trinucleotide repeats: a novel architecture of RNA quadruplexes

    PubMed Central

    Gudanis, Dorota; Popenda, Lukasz; Szpotkowski, Kamil; Kierzek, Ryszard; Gdaniec, Zofia

    2016-01-01

    Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a ‘toxic’ gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine (BrG)-modified molecule GCBrGGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GCBrGGCGGC)4 RNA quadruplex with BrG3:G6:BrG3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson–Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry. PMID:26743003

  15. Structural characterization of a dimer of RNA duplexes composed of 8-bromoguanosine modified CGG trinucleotide repeats: a novel architecture of RNA quadruplexes.

    PubMed

    Gudanis, Dorota; Popenda, Lukasz; Szpotkowski, Kamil; Kierzek, Ryszard; Gdaniec, Zofia

    2016-03-18

    Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a 'toxic' gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine ((Br)G)-modified molecule GC(Br)GGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GC(Br)GGCGGC)4 RNA quadruplex with (Br)G3:G6:(Br)G3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson-Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry. PMID:26743003

  16. Label-free microRNA detection based on terbium and duplex-specific nuclease assisted target recycling.

    PubMed

    Zhang, Jing; Wu, Dongzhi; Chen, QiuXiang; Chen, Mei; Xia, Yaokun; Cai, Shuxian; Zhang, Xi; Wu, Fang; Chen, Jinghua

    2015-08-01

    In this paper, we describe a novel label-free fluorescence method for microRNA-21 (miR-21) detection based on terbium (Tb(3+)) and duplex-specific nuclease (DSN) assisted target recycling. Capture probes (Cps), containing a target-binding part and a signal-output part, are immobilized on magnetic beads (MBs). In the presence of the target miR-21, it hybridizes with the target-binding part of a Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzes the target-binding part of the Cp while liberating the intact target miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually, through magnetic separation, only the signal-output part of the Cp could remain in solution and function as a signalling flare to increase the fluorescence intensity of Tb(3+) dramatically. By employing the above strategy, this approach can gain an amplified fluorescent signal and detect as low as 8 fM miR-21 under the optimized conditions. Moreover, due to the high selectivity of DSN, the method shows little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human cell lysate samples of breast cancer patients. PMID:26106867

  17. Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site.

    PubMed

    Ennifar, Eric; Walter, Philippe; Dumas, Philippe

    2010-09-01

    The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3'-phosphate and 5'-hydroxyl termini. A crystallographic analysis showed that Ba(2+), Mn(2+), Co(2+) and Zn(2+) bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an 'in-line' geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a 'Trojan horse' mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a 'harmless' species is further transformed in situ into an 'aggressive' species carrying a hydroxide anion. PMID:20460458

  18. Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

    PubMed Central

    Ennifar, Eric; Walter, Philippe; Dumas, Philippe

    2010-01-01

    The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba2+, Mn2+, Co2+ and Zn2+ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion. PMID:20460458

  19. Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR.

    PubMed

    Barros, Silvia C; Ramos, Fernanda; Zé-Zé, Líbia; Alves, Maria J; Fagulha, Teresa; Duarte, Margarida; Henriques, Margarida; Luís, Tiago; Fevereiro, Miguel

    2013-11-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies. PMID:23892127

  20. Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex

    PubMed Central

    Plevnik, Miha; Gdaniec, Zofia; Plavec, Janez

    2005-01-01

    The isomerization of phosphodiester functionality of nucleic acids from 3′,5′- to a less common 2′,5′-linkage influences the complex interplay of stereoelectronic effects that drive pseudorotational equilibrium of sugar rings and thus affect the conformational propensities for compact or more extended structures. The present study highlights the subtle balance of non-covalent forces at play in structural equilibrium of 2′,5′-linked RNA analogue, 3′-O-(2-methoxyethyl) substituted dodecamer *CG*CGAA*U*U*CG*CG, 3′-MOE-2′,5′-RNA, where all cytosines and uracils are methylated at C5. The NMR and UV spectroscopic studies have shown that 3′-MOE-2′,5′-RNA adopts both hairpin and duplex secondary structures, which are involved in a dynamic exchange that is slow on the NMR timescale and exhibits strand and salt concentration as well as pH dependence. Unusual effect of pH over a narrow physiological range is observed for imino proton resonances with exchange broadening observed at lower pH and relatively sharp lines observed at higher pH. The solution structure of 3′-MOE-2′,5′-RNA hairpin displays a unique and well-defined loop, which is stabilized by Watson–Crick A5·*U8 base pair and by n → π* stacking interactions of O4′ lone-pair electrons of A6 and *U8 with aromatic rings of A5 and *U7, respectively. In contrast, the stem region of 3′-MOE-2′,5′-RNA hairpin is more flexible. Our data highlight the important feature of backbone modifications that can have pronounced effects on interstrand association of nucleic acids. PMID:15788747

  1. Increasing the Stability of DNA:RNA Duplexes by Introducing Stacking Phenyl-Substituted Pyrazole, Furan, and Triazole Moieties in the Major Groove.

    PubMed

    Hornum, Mick; Kumar, Pawan; Podsiadly, Patricia; Nielsen, Poul

    2015-10-01

    Consecutive incorporations of our previously published thymidine analogue, 5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W in oligonucleotides, has demonstrated significant duplex-stabilizing properties due to its efficient staking properties in the major groove of DNA:RNA duplexes. The corresponding 2'-deoxycytidine analogue is not as well-accommodated in duplexes, however, due to its clear preference for the ring-flipped coplanar conformation. In our present work, we have used ab initio calculations to design two new building blocks, 5-(5-phenylfuran-2-yl)-2'-deoxycytidine monomer Y and 5-(1-phenyl-1H-pyrazol-3-yl)-2'-deoxycytidine monomer Z, that emulate the conformation of W. These monomers were synthesized by Suzuki-Miyaura couplings, and the pyrazole moiety was obtained in a cycloaddition from N-phenylsydnone. We show that the novel analogues Y and Z engage in efficient stacking either with themselves or with W due to a better overlap of the aromatic moieties. Importantly, we demonstrate that this translates into very thermally stable DNA:RNA duplexes, thus making Y and especially Z good candidates for improving the binding affinities of oligonucleotide-based therapeutics. Since we now have both efficiently stacking T and C analogues in hand, any purine rich stretch can be effectively targeted using these simple analogues. Notably, we show that the introduction of the aromatic rings in the major groove does not significantly change the helical geometry. PMID:26334359

  2. Fe₃O₄@Ag magnetic nanoparticles for microRNA capture and duplex-specific nuclease signal amplification based SERS detection in cancer cells.

    PubMed

    Pang, Yuanfeng; Wang, Chongwen; Wang, Jing; Sun, Zhiwei; Xiao, Rui; Wang, Shengqi

    2016-05-15

    A functionalized Fe3O4@Ag magnetic nanoparticle (NP) biosensor for microRNA (miRNA) capture and ultrasensitive detection in total RNA extract from cancer cells was reported in this paper. Herein, Raman tags-DNA probes modified Fe3O4@Ag NPs were designed both as surface-enhanced Raman scattering (SERS) SERS and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, target miRNAs were captured to the surface of Fe3O4@Ag NPs through DNA/RNA hybridization. In the presence of endonuclease duplex specific nuclease (DSN), one target miRNA molecule could rehybrid thousands of DNA probes to trigger the signal-amplifying recycling. Base on the superparamagnetic of Fe3O4@Ag NPs, target miRNA let-7b can be captured, concentrated and direct quantified within a PE tube without any PCR preamplification treatment. The detection limit was 0.3fM (15 zeptomole, 50μL), nearly 3 orders of magnitude lower than conventional fluorescence based DSN biosensors for miRNA(∼100fM), even single-base difference between the let-7 family members can be discriminated. The result provides a novel proposal to combine the perfect single-base recognition and signal-amplifying ability of the endonuclease DSN with cost-effective SERS strategy for miRNA point-of-care (POC) clinical diagnostics. PMID:26749099

  3. G-Quadruplex Folds of the Human Telomere Sequence Alter the Site Reactivity and Reaction Pathway of Guanine Oxidation Compared to Duplex DNA

    PubMed Central

    Fleming, Aaron M.; Burrows, Cynthia J.

    2013-01-01

    Telomere shortening occurs during oxidative and inflammatory stress with guanine (G) as the major site of damage. In this work, a comprehensive profile of the sites of oxidation and structures of products observed from G-quadruplex and duplex structures of the human telomere sequence was studied in the G-quadruplex folds (hybrid (K+), basket (Na+), and propeller (K+ + 50% CH3CN)) resulting from the sequence 5’-(TAGGGT)4T-3’ and in an appropriate duplex containing one telomere repeat. Oxidations with four oxidant systems consisting of riboflavin photosensitization, carbonate radical generation, singlet oxygen, and the copper Fenton-like reaction were analyzed under conditions of low product conversion to determine relative reactivity. The one-electron oxidants damaged the 5’-G in G-quadruplexes leading to spiroiminodihydantoin (Sp) and 2,2,4-triamino-2H-oxazol-5-one (Z) as major products as well as 8-oxo-7,8-dihydroguanine (OG) and 5-guanidinohydantoin (Gh) in low relative yields, while oxidation in the duplex context produced damage at the 5’- and middle-Gs of GGG sequences and resulted in Gh being the major product. Addition of the reductant N-acetylcysteine (NAC) to the reaction did not alter the riboflavin-mediated damage sites, but decreased Z by 2-fold and increased OG by 5-fold, while not altering the hydantoin ratio. However, NAC completely quenched the CO3•− reactions. Singlet oxygen oxidations of the G-quadruplex showed reactivity at all Gs on the exterior faces of G-quartets and furnished the product Sp, while no oxidation was observed in the duplex context under these conditions, and addition of NAC had no effect. Because a long telomere sequence would have higher-order structures of G-quadruplexes, studies were also conducted with 5’-(TAGGGT)8-T-3’, and it provided similar oxidation profiles to the single G-quadruplex. Lastly, CuII/H2O2-mediated oxidations were found to be indiscriminate in the damage patterns, and 5-carboxamido-5

  4. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    PubMed Central

    Ahlenstiel, Chantelle; Mendez, Catalina; Lim, Steven T H; Marks, Katherine; Turville, Stuart; Cooper, David A; Kelleher, Anthony D; Suzuki, Kazuo

    2015-01-01

    Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation. PMID:26506039

  5. The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate.

    PubMed

    Mooers, Blaine H M; Singh, Amritanshu

    2011-10-01

    Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 5' end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 3' ends of most guide RNAs have ∼15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G·U wobble base pairs. To gain insights into the structure of the U-helix, we crystallized 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 5'-AGA-3'/5'-UUU-3' base-pair triple. The fusion of two identical U-helices head-to-head promoted crystallization. We obtained X-ray diffraction data with a resolution limit of 1.37 Å. The U-helix had low and high twist angles before and after each G·U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins. PMID:21878548

  6. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex

    SciTech Connect

    Wang, Yanli; Juranek, Stefan; Li, Haitao; Sheng, Gang; Tuschl, Thomas; Patel, Dinshaw J.

    2009-01-08

    Here we report on a 3.0 {angstrom} crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.

  7. X-ray Structures of U2 snRNA-Branchpoint Duplexes Containing Conserved Pseudouridines

    SciTech Connect

    Lin,Y.; Kielkopf, C.

    2008-01-01

    A pseudouridine-modified region of the U2 small nuclear (sn)RNA anneals with the intronic branchpoint sequence and positions a bulged adenosine to serve as the nucleophile in the first chemical step of pre-mRNA splicing. We have determined three X-ray structures of RNA oligonucleotides containing the pseudouridylated U2 snRNA and the branchpoint consensus sequences. The expected adenosine branchpoint is extrahelical in a 1.65 Angstroms resolution structure containing the mammalian consensus sequence variant and in a 2.10 Angstroms resolution structure containing a shortened Saccharomyces cerevisiae consensus sequence. The adenosine adjacent to the expected branchpoint is extrahelical in a third structure, which contains the intact yeast consensus sequence at 1.57 Angstroms resolution. The hydration and base stacking interactions mediated by the U2 snRNA pseudouridines correlate with the identity of the unpaired adenosine. The expected adenosine bulge is associated with a well-stacked pseudouridine, which is linked via an ordered water molecule to a neighboring nucleotide. In contrast, the bulge of the adjacent adenosine shifts the base stacking and disrupts the water-mediated interactions of the pseudouridine. These structural differences may contribute to the ability of the pseudouridine modification to promote the bulged conformation of the branch site adenosine and to enhance catalysis by snRNAs. Furthermore, iodide binding sites are identified adjacent to the unconventional bulged adenosine, and the structure of the mammalian consensus sequence variant provides a high-resolution view of a hydrated magnesium ion bound in a similar manner to a divalent cation binding site of the group II intron.

  8. Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study.

    PubMed

    Pan, Feng; Roland, Christopher; Sagui, Celeste

    2014-12-16

    The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5'-CGCGCGCGCGCG-3')2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na(+), K(+) and Mg(2 +), with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6](2+) ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6](2+) to distant phosphates, resulting in acute bending of A-RNA; tight 'ion traps' in Z-RNA between C-O2 and the C-O2' atoms in GpC steps; and others. PMID:25428372

  9. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2. PMID:26876600

  10. Atomic resolution structure of a chimeric DNA-RNA Z-type duplex in complex with Ba(2+) ions: a case of complicated multi-domain twinning.

    PubMed

    Gilski, Miroslaw; Drozdzal, Pawel; Kierzek, Ryszard; Jaskolski, Mariusz

    2016-02-01

    The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of the P21 crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba(2+) sites. The chimeric (DNA-RNA)2 duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA-RNA chimeric duplex in complex with Ba(2+) ions and also with Sr(2+) ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groups P1 and P21 but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space group P21 to an R and Rfree of 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases. PMID:26894669

  11. Synthesis of new C-5-triazolyl-functionalized thymidine analogs and their ability to engage in aromatic stacking in DNA : DNA and DNA : RNA duplexes.

    PubMed

    Hornum, Mick; Djukina, Alevtina; Sassnau, Ann-Katrin; Nielsen, Poul

    2016-05-11

    1-Phenyl-1,2,3-triazole scaffolds on the 5-position of pyrimidine nucleosides have previously shown to enhance nuclease stability and increase the duplex thermal stability (Tm) by engaging in duplex stacking interactions. In this study, we have introduced two new derivatives of this scaffold in DNA : DNA and DNA : RNA duplexes in order to explore the thermal effects of (1) using a 1,5-triazole instead of the usual 1,4-triazole, and (2) replacing the apolar phenyl substituent with a polar uracil-5-yl substituent. PMID:27089052

  12. Airway Epithelial miRNA Expression Is Altered in Asthma

    PubMed Central

    Solberg, Owen D.; Ostrin, Edwin J.; Love, Michael I.; Peng, Jeffrey C.; Bhakta, Nirav R.; Nguyen, Christine; Solon, Margaret; Nguyen, Cindy; Barczak, Andrea J.; Zlock, Lorna T.; Blagev, Denitza P.; Finkbeiner, Walter E.; Ansel, K. Mark; Arron, Joseph R.; Erle, David J.

    2012-01-01

    Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13–regulated miRNAs. Methods: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway

  13. Duplex ultrasound

    MedlinePlus

    Vascular ultrasound; Peripheral vascular ultrasound ... A duplex ultrasound combines traditional ultrasound with Doppler ultrasound . Traditional ultrasound uses sound waves that bounce off blood vessels to create ...

  14. Recognition of mixed-sequence DNA duplexes: Design guidelines for Invaders based on 2′-O-(pyren-1-yl)methyl-RNA monomers

    PubMed Central

    Karmakar, Saswata; Guenther, Dale C.; Hrdlicka*, Patrick J.

    2013-01-01

    Development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable due to their potential as tools for detection, regulation and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-O-(pyren-1-yl)methyl-RNA monomers and have been recently shown to recognize linear dsDNA, DNA hairpins and chromosomal DNA. In the present work, we systematically study the role that the nucleobase moieties of the 2′-O-(pyren-1-yl)methyl-RNA monomers have on recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with +1 interstrand zippers of the four canonical 2′-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers, show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprised of C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of effective Invaders for applications in molecular biology, nucleic acid diagnostics and biotechnology. PMID:24195730

  15. An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification.

    PubMed Central

    Blommers, M J; Pieles, U; De Mesmaeker, A

    1994-01-01

    The backbone modification amide-3, in which -CH2-NH-CO-CH2- replaces -C5'H2-O5'-PO2-O3'-, is studied in the duplex d(G1-C2-G3-T4.T5-G6-C7-G8)*mr(C9-G10-C11-A12-A13-C14-G15+ ++-C16) where . indicates the backbone modification and mr indicates the 2'-OMe RNA strand. The majority of the exchangeable and non-exchangeable resonances have been assigned. The assignment procedure differs from standard methods. The methyl substituent of the 2'-OMe position of the RNA strand can be used as a tool in the interpretation. The duplex structure is a right-handed double helix. The sugar conformations of the 2'-OMe RNA strand are predominantly N-type and the 2'-OMe is positioned at the surface of the minor groove. In the complementary strand, only the sugar of residue T4 is found exclusively in N-type conformation. The incorporation of the amide modification does not effect very strongly the duplex structure. All bases are involved in Watson-Crick base pairs. PMID:7524037

  16. Phosphorus-31 NMR spectra of ethidium, quinacrine, and daunomycin complexes with poly(adenylic acid)ter dot poly(uridylic acid) RNA duplex and calf thymus DNA

    SciTech Connect

    Gorenstein, D.G.; Lai, K. )

    1989-04-04

    {sup 31}P NMR provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the intercalating drugs ethidium, quinacrine, and daunomycin to sonicated poly(A){center dot}poly(U) and calf thymus DNA. {sup 31}P chemical shifts can also be used to assess differences in the duplex unwinding angles in the presence of the drug. Thus a new {sup 31}P signal, 1.8-2.2 ppm downfield from the double-stranded helix signals, is observed in the ethidium ion-poly(A){center dot}poly(U) complex. This signal arises from phosphates which are in perturbed environments due to intercalation of the drug. This is in keeping with the hypothesis that the P-O ester torsional angle in phosphates linking the intercalated base pairs is more trans-like. Similar though smaller deshielding of the {sup 31}P signals is observed in sonicated poly(A){center dot}poly(U)-quinacrine complexes as well as in the daunomycin complexes. The effect of added ethidium ion, quinacrine, and daunomycin on the {sup 31}P spectra of sonicated calf thymus DNA is consistent with Wilson and Jones' (1982) earlier study. In these drug-DNA complexes the drug produces a gradual downfield shift in the DNA {sup 31}P signal without the appearance of a separate downfield peak. These differences are attributed to differences in the rate of chemical exchange of the drug between free and bound duplex states. The previous correlation of {sup 31}P chemical shift with drug duplex unwinding angle is confirmed for both the RNA and DNA duplexes.

  17. A novel polydopamine-based chemiluminescence resonance energy transfer method for microRNA detection coupling duplex-specific nuclease-aided target recycling strategy.

    PubMed

    Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

    2016-06-15

    MicroRNAs (miRNAs), functioning as oncogenes or tumor suppressors, play significant regulatory roles in regulating gene expression and become as biomarkers for disease diagnostics and therapeutics. In this work, we have coupled a polydopamine (PDA) nanosphere-assisted chemiluminescence resonance energy transfer (CRET) platform and a duplex-specific nuclease (DSN)-assisted signal amplification strategy to develop a novel method for specific miRNA detection. With the assistance of hemin, luminol, and H2O2, the horseradish peroxidase (HRP)-mimicking G-rich sequence in the sensing probe produces chemiluminescence, which is quickly quenched by the CRET effect between PDA as energy acceptor and excited luminol as energy donor. The target miRNA triggers DSN to partially degrade the sensing probe in the DNA-miRNA heteroduplex to repeatedly release G-quadruplex formed by G-rich sequence from PDA for the production of chemiluminescence. The method allows quantitative detection of target miRNA in the range of 80 pM-50 nM with a detection limit of 49.6 pM. The method also shows excellent specificity to discriminate single-base differences, and can accurately quantify miRNA in biological samples, with good agreement with the result from a commercial miRNA detection kit. The procedure requires no organic dyes or labels, and is a simple and cost-effective method for miRNA detection for early clinical diagnosis. PMID:26866561

  18. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  19. Study of E. coli Hfq’s RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

    PubMed Central

    Doetsch, Martina; Stampfl, Sabine; Fürtig, Boris; Beich-Frandsen, Mads; Saxena, Krishna; Lybecker, Meghan; Schroeder, Renée

    2013-01-01

    Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other. PMID:23104381

  20. An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA.

    PubMed

    Zhang, Jing; Wu, Dong-Zhi; Cai, Shu-Xian; Chen, Mei; Xia, Yao-Kun; Wu, Fang; Chen, Jing-Hua

    2016-01-15

    An immobilization-free electrochemical impedance biosensor for microRNA detection was developed in this work, which was based on both the duplex-specific nuclease assisted target recycling (DSNATR) and capture probes (Cps) enriched from the solution to electrode surface via magnetic beads (MBs). In the absence of miR-21, Cps cannot be hydrolyzed due to the low activity of duplex-specific nuclease (DSN) against ssDNA. Therefore, the intact Cps could be attached to the surface of magnetic glass carbon electrode (MGCE), resulting in a compact negatively charged layer as well as a large charge-transfer resistance. While in the presence of miR-21, it hybridized with Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzed the target-binding part of the Cp while liberating the intact miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. In this way, a single miR-21 was able to trigger the permanent hydrolysis of multiple Cps. Finally, all Cps were digested. Thus, the negatively charged layer could not be formed, resulting in a small charge-transfer resistance. By employing the above strategy, the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with a detection limit of 60aM. Meanwhile, the method showed little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human serum samples of breast cancer patients. PMID:26363493

  1. Substrate recognition and specificity of double-stranded RNA binding proteins.

    PubMed

    Vuković, Lela; Koh, Hye Ran; Myong, Sua; Schulten, Klaus

    2014-06-01

    Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins. PMID:24801449

  2. MicroRNA-Offset RNA Alters Gene Expression and Cell Proliferation

    PubMed Central

    Zhao, Jin; Schnitzler, Gavin R.; Iyer, Lakshmanan K.; Aronovitz, Mark J.; Baur, Wendy E.; Karas, Richard H.

    2016-01-01

    MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a “seed region” of moR-21 as well as a “seed match region” in the target gene 3’UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active. PMID:27276022

  3. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

    PubMed Central

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (Kd ∼ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (Kd ∼ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA. PMID:17942426

  4. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

    PubMed

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA. PMID:17942426

  5. Altered RNA editing in 3′ UTR perturbs microRNA-mediated regulation of oncogenes and tumor-suppressors

    PubMed Central

    Zhang, Liye; Yang, Chih-Sheng; Varelas, Xaralabos; Monti, Stefano

    2016-01-01

    RNA editing is a molecular event that alters specific nucleotides in RNA post-transcriptionally. RNA editing has the potential to impact a variety of cellular processes and is implicated in diseases such as cancer. Yet, the precise mechanisms by which RNA editing controls cellular processes are poorly understood. Here, we characterize sequences altered by RNA editing in patient samples from lymphoma, neuroblastoma and head and neck cancers. We show that A-to-I RNA editing sites are highly conserved across samples of the same tissue type and that most editing sites identified in tumors are also detectable in normal tissues. Next, we identify the significant changes in editing levels of known sites between tumor and paired “normal” tissues across 14 cancer types (627 pairs) from The Cancer Genome Atlas project and show that the complexity of RNA editing regulation cannot be captured by the activity of ADAR family genes alone. Our pan-cancer analysis confirms previous results on individual tumor types and suggests that changes of RNA editing levels in coding and 3′UTR regions could be a general mechanism to promote tumor growth. We also propose a model explaining how altered RNA editing levels affect microRNA-mediated post-transcriptional regulation of oncogenes and tumor-suppressors. PMID:26980570

  6. Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution.

    PubMed Central

    Lima, Susana; Hildenbrand, Jayne; Korostelev, Andrei; Hattman, Stanley; Li, Hong

    2002-01-01

    The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com. PMID:12166647

  7. SomamiR 2.0: a database of cancer somatic mutations altering microRNA-ceRNA interactions.

    PubMed

    Bhattacharya, Anindya; Cui, Yan

    2016-01-01

    SomamiR 2.0 (http://compbio.uthsc.edu/SomamiR) is a database of cancer somatic mutations in microRNAs (miRNA) and their target sites that potentially alter the interactions between miRNAs and competing endogenous RNAs (ceRNA) including mRNAs, circular RNAs (circRNA) and long noncoding RNAs (lncRNA). Here, we describe the recent major updates to the SomamiR database. We expanded the scope of the database by including somatic mutations that impact the interactions between miRNAs and two classes of non-coding RNAs, circRNAs and lncRNAs. Recently, a large number of miRNA target sites have been discovered by newly emerged high-throughput technologies for mapping the miRNA interactome. We have mapped 388 247 somatic mutations to the experimentally identified miRNA target sites. The updated database also includes a list of somatic mutations in the miRNA seed regions, which contain the most important guiding information for miRNA target recognition. A recently developed webserver, miR2GO, was integrated with the database to provide a seamless pipeline for assessing functional impacts of somatic mutations in miRNA seed regions. Data and functions from multiple sources including biological pathways and genome-wide association studies were updated and integrated with SomamiR 2.0 to make it a better platform for functional analysis of somatic mutations altering miRNA-ceRNA interactions. PMID:26578591

  8. Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

    PubMed

    Rupaimoole, Rajesha; Lee, Jaehyuk; Haemmerle, Monika; Ling, Hui; Previs, Rebecca A; Pradeep, Sunila; Wu, Sherry Y; Ivan, Cristina; Ferracin, Manuela; Dennison, Jennifer B; Millward, Niki M Zacharias; Nagaraja, Archana S; Gharpure, Kshipra M; McGuire, Michael; Sam, Nidhin; Armaiz-Pena, Guillermo N; Sadaoui, Nouara C; Rodriguez-Aguayo, Cristian; Calin, George A; Drapkin, Ronny I; Kovacs, Jeffery; Mills, Gordon B; Zhang, Wei; Lopez-Berestein, Gabriel; Bhattacharya, Pratip K; Sood, Anil K

    2015-12-22

    Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer. PMID:26686630

  9. Long noncoding RNA ceruloplasmin promotes cancer growth by altering glycolysis

    PubMed Central

    Rupaimoole, Rajesha; Lee, Jaehyuk; Haemmerle, Monika; Ling, Hui; Previs, Rebecca A.; Pradeep, Sunila; Wu, Sherry Y.; Ivan, Cristina; Ferracin, Manuela; Dennison, Jennifer B.; Zacharias Millward, Niki M.; Nagaraja, Archana S.; Gharpure, Kshipra M.; McGuire, Michael; Sam, Nidhin; Armaiz-Pena, Guillermo N.; Sadaoui, Nouara C.; Rodriguez-Aguayo, Cristian; Calin, George A.; Drapkin, Ronny I.; Kovacs, Jeffery; Mills, Gordon B.; Zhang, Wei; Lopez-Berestein, Gabriel; Bhattacharya, Pratip K.; Sood, Anil K.

    2015-01-01

    SUMMARY Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue towards modulating lncRNAs in cancer. PMID:26686630

  10. DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition

    PubMed Central

    Samatanga, Brighton; Klostermeier, Dagmar

    2014-01-01

    DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes. PMID:25123660

  11. Acquired copy number alterations of miRNA genes in acute myeloid leukemia are uncommon

    PubMed Central

    Ramsingh, Giridharan; Jacoby, Meagan A.; Shao, Jin; De Jesus Pizzaro, Rigoberto E.; Shen, Dong; Trissal, Maria; Getz, Angela H.; Ley, Timothy J.; Walter, Matthew J.

    2013-01-01

    Altered microRNA (miRNA) expression is frequently observed in acute myelogenous leukemia (AML) and has been implicated in leukemic transformation. Whether somatic copy number alterations (CNAs) are a frequent cause of altered miRNA gene expression is largely unknown. Herein, we used comparative genomic hybridization with a custom high-resolution miRNA-centric array and/or whole-genome sequence data to identify somatic CNAs involving miRNA genes in 113 cases of AML, including 50 cases of de novo AML, 18 cases of relapsed AML, 15 cases of secondary AML following myelodysplastic syndrome, and 30 cases of therapy-related AML. We identified a total of 48 somatic miRNA gene-containing CNAs that were not identified by routine cytogenetics in 20 patients (18%). All these CNAs also included one or more protein coding genes. We identified a single case with a hemizygous deletion of MIR223, resulting in the complete loss of miR-223 expression. Three other cases of AML were identified with very low to absent miR-223 expression, an miRNA gene known to play a key role in myelopoiesis. However, in these cases, no somatic genetic alteration of MIR223 was identified, suggesting epigenetic silencing. These data show that somatic CNAs specifically targeting miRNA genes are uncommon in AML. PMID:24009227

  12. Altered A-to-I RNA editing in human embryogenesis.

    PubMed

    Shtrichman, Ronit; Germanguz, Igal; Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  13. Altered A-to-I RNA Editing in Human Embryogenesis

    PubMed Central

    Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  14. Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

    PubMed Central

    Shirai, Cara Lunn; Ley, James N.; White, Brian S.; Kim, Sanghyun; Tibbitts, Justin; Shao, Jin; Ndonwi, Matthew; Wadugu, Brian; Duncavage, Eric J.; Okeyo-Owuor, Theresa; Liu, Tuoen; Griffith, Malachi; McGrath, Sean; Magrini, Vincent; Fulton, Robert S.; Fronick, Catrina; O’Laughlin, Michelle; Graubert, Timothy A.; Walter, Matthew J.

    2015-01-01

    SUMMARY Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ~11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently-mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in MDS patients. PMID:25965570

  15. Structural alterations of the ribosomal RNA genes in leukemic cells.

    PubMed

    Smirnova, I A

    1992-01-01

    Cloned 6.7 kb EcoR1 fragment of mice rDNA was used as a hybridization probe for rDNA structure analysis in mice, rat and calf haemopoietic tumor and normal cells. EcoR1, BglII and Pst1 restriction fragment length polymorphism (RFLP) was found in neoplastic rDNA and was not revealed in normal ones. The rRNA gene rearrangements were observed not only in spacer region but in coding sequences of the genes. Leukemic cells reveal also rDNA amplification. A role of genetic rearrangements of rDNA for mechanisms of carcinogenesis is suggested. PMID:1342066

  16. Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes.

    PubMed

    Hansen, Allison K; Moran, Nancy A

    2012-09-01

    Translational efficiency is controlled by tRNAs and other genome-encoded mechanisms. In organelles, translational processes are dramatically altered because of genome shrinkage and horizontal acquisition of gene products. The influence of genome reduction on translation in endosymbionts is largely unknown. Here, we investigate whether divergent lineages of Buchnera aphidicola, the reduced-genome bacterial endosymbiont of aphids, possess altered translational features compared with their free-living relative, Escherichia coli. Our RNAseq data support the hypothesis that translation is less optimal in Buchnera than in E. coli. We observed a specific, convergent, pattern of tRNA loss in Buchnera and other endosymbionts that have undergone genome shrinkage. Furthermore, many modified nucleoside pathways that are important for E. coli translation are lost in Buchnera. Additionally, Buchnera's A + T compositional bias has resulted in reduced tRNA thermostability, and may have altered aminoacyl-tRNA synthetase recognition sites. Buchnera tRNA genes are shorter than those of E. coli, as the majority no longer has a genome-encoded 3' CCA; however, all the expressed, shortened tRNAs undergo 3' CCA maturation. Moreover, expression of tRNA isoacceptors was not correlated with the usage of corresponding codons. Overall, our data suggest that endosymbiont genome evolution alters tRNA characteristics that are known to influence translational efficiency in their free-living relative. PMID:22689638

  17. New sensitive one-step real-time duplex PCR method for group A and B HIV-2 RNA load.

    PubMed

    Avettand-Fenoel, Véronique; Damond, Florence; Gueudin, Marie; Matheron, Sophie; Mélard, Adeline; Collin, Gilles; Descamps, Diane; Chaix, Marie-Laure; Rouzioux, Christine; Plantier, Jean-Christophe

    2014-08-01

    The Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) previously developed a widely used method for HIV-1 RNA quantification (Biocentric). Here, we report the development of a new specific and sensitive method for HIV-2 RNA quantification, based on an adaptation of the existing HIV-1 protocol. The new test is based on TaqMan one-step reverse transcription-quantitative PCR (qRT-PCR) targeting two conserved consensus regions of HIV-2 (long terminal repeat [LTR] and gag). Analytic performances were determined in three laboratories. Clinical performances were evaluated on 100 plasma samples from HIV-2-infected patients (groups A, B, and H) by comparison with the assay currently used for the ANRS HIV-2 cohort. The specificity was 100%. Sensitivity was 50 copies/ml (cp/ml) and was optimized to 10 cp/ml. The within-run coefficients of variation in the three laboratories varied from 0.54% to 1.61% at 4 log10 copies/ml and from 7.24% to 14.32% at 2 log10 cp/ml. The between-run coefficients of variation varied from 2.28% to 6.43%. Of the 39 clinical samples below 2 log10 in the current assay, the new test improved the detection or quantification of 17 samples, including eight group B samples. For quantifiable samples, similar loads were obtained with the two assays for group A samples. The median difference between the two assays for group B samples was +0.18 but with greater heterogeneity than for group A. The HIV-2 group H sample had similar results with the two assays. This new assay is highly sensitive and accurately quantifies the most prevalent HIV-2 groups. This test will be useful for monitoring low viral loads in HIV-2-infected patients. PMID:24920771

  18. Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates.

    PubMed

    Albiach-Martí, M R; Guerri, J; de Mendoza, A H; Laigret, F; Ballester-Olmos, J F; Moreno, P

    2000-02-01

    ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates. PMID:18944601

  19. Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression

    PubMed Central

    Yu, Mengying; Wu, Haibo; Ai, Zhiying; Wu, Yongyan; Liu, Hongliang; Du, Juan; Guo, Zekun; Zhang, Yong

    2015-01-01

    Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs. PMID:26162091

  20. Formaldehyde exposure alters miRNA expression profiles in the olfactory bulb.

    PubMed

    Li, Guifa; Yang, Jing; Ling, Shucai

    2015-01-01

    It has been reported that inhaling formaldehyde (FA) causes damage to the central nervous system. However, it is unclear whether FA can disturb the function of the olfactory bulb. Using a microarray, we found that FA inhalation altered the miRNA expression profile. Functional enrichment analysis of the predicted targets of the changed miRNA showed that the enrichment canonical pathways and networks associated with cancer and transcriptional regulation. FA exposure disrupts miRNA expression profiles within the olfactory bulb. PMID:26161908

  1. Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain.

    PubMed

    Richardson, Charles J; First, Eric A

    2016-03-15

    Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine. PMID:26890980

  2. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    PubMed

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. PMID:25881991

  3. Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts

    PubMed Central

    Cross, Courtney E.; Tolba, Mai F.; Rondelli, Catherine M.; Xu, Meixiang; Abdel-Rahman, Sherif Z.

    2015-01-01

    The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development. PMID:26339600

  4. Polymicrobial infection alter inflammatory microRNA in rat salivary glands during periodontal disease.

    PubMed

    Nayar, Gautam; Gauna, Adrienne; Chukkapalli, Sasanka; Velsko, Irina; Kesavalu, Lakshmyya; Cha, Seunghee

    2016-04-01

    Periodontal disease initiated by subgingival pathogens is linked with diminished secretion of saliva, and implies pathogenic bacteria dissemination to or affects secondary sites such as the salivary glands. MicroRNAs activated in response to bacteria may modulate immune responses against pathogens. Therefore, Sprague-Dawley rats were infected by oral lavage consisting of polymicrobial inocula, namely Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, or sham-infected for 12 weeks (n = 6). We quantified inflammatory miRNA expression levels of miRNA-132, miR-146a, and miR-155 at secondary sites to the primary infection of the gingiva, including submandibular salivary glands, lacrimal glands, and pancreas. The presence of bacteria was detected in situ at secondary sites. Infected rat gingiva showed increased relative expression of miR-155. In contrast, miRNA-155 expression was decreased in submandibular salivary glands, along with positive identification of P. gingivalis in 2/6 and T. denticola in 1/6 rat salivary glands. Furthermore, miRNA-132 and miRNA-146a were significantly decreased in the pancreas of infected rats. This study is the first to show primary periodontal infections can alter miRNA profiles in secondary sites such as the salivary gland and pancreas. Whether these alterations contribute to pathologies of salivary glands in Sjögren's syndrome or of pancreas in diabetes warrants further investigation. PMID:26481834

  5. Alterations in miRNA Levels in the Dentate Gyrus in Epileptic Rats

    PubMed Central

    Bot, Anna Maria; Dębski, Konrad Józef; Lukasiuk, Katarzyna

    2013-01-01

    The aim of this study was to characterize changes in miRNA expression in the epileptic dentate gyrus. Status epilepticus evoked by amygdala stimulation was used to induce epilepsy in rats. The dentate gyri were isolated at 7 d, 14 d, 30 d and 90 d after stimulation (n=5). Sham-operated time-matched controls were prepared for each time point (n=5). The miRNA expression was evaluated using Exiqon microarrays. Additionally, mRNA from the same animals was profiled using Affymetrix microarrays. We detected miRNA expression signatures that differentiate between control and epileptic animals. Significant changes in miRNA expression between stimulated and sham operated animals were observed at 7 and 30 d following stimulation. Moreover, we found that there are ensembles of miRNAs that change expression levels over time. Analysis of the mRNA expression from the same animals revealed that the expression of several mRNAs that are potential targets for miRNA with altered expression level is regulated in the expected direction. The functional characterization of miRNAs and their potential mRNA targets indicate that miRNA can participate in several molecular events that occur in epileptic tissue, including immune response and neuronal plasticity. This is the first report on changes in the expression of miRNA and the potential functional impact of these changes in the dentate gyrus of epileptic animals. Complex changes in the expression of miRNAs suggest an important role for miRNA in the molecular mechanisms of epilepsy. PMID:24146813

  6. MicroRNA Expression Signature Is Altered in the Cardiac Remodeling Induced by High Fat Diets.

    PubMed

    Guedes, Elaine Castilho; França, Gustavo Starvaggi; Lino, Caroline Antunes; Koyama, Fernanda Christtanini; Moreira, Luana do Nascimento; Alexandre, Juliana Gomes; Barreto-Chaves, Maria Luiza M; Galante, Pedro Alexandre Favoretto; Diniz, Gabriela Placoná

    2016-08-01

    Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the cardiac miRNA expression signature. To address this question, male C57Bl/6 mice were fed with a low fat (LF) diet or two HF diets, containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides, and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart. HF45% diet for 10 and 20 weeks changed the abundance of 64 and 26 miRNAs in the heart, respectively. On the other hand, HF60% diet for 10 and 20 weeks altered the abundance of 27 and 88 miRNAs in the heart, respectively. Bioinformatics analysis indicated that insulin signaling pathway was overrepresented in response to HF diet. An inverse correlation was observed between cardiac levels of GLUT4 and miRNA-29c. Similarly, we found an inverse correlation between expression of GSK3β and the expression of miRNA-21a-3p, miRNA-29c-3p, miRNA-144-3p, and miRNA-195a-3p. In addition, miRNA-1 overexpression prevented cardiomyocyte hypertrophy. Taken together, our results revealed differentially expressed miRNA signatures in the heart in response to different HF diets. J. Cell. Physiol. 231: 1771-1783, 2016. © 2015 Wiley Periodicals, Inc. PMID:26638879

  7. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  8. 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

  9. Smoking alters circulating plasma microvesicle pattern and microRNA signatures.

    PubMed

    Badrnya, S; Baumgartner, R; Assinger, A

    2014-07-01

    Circulating plasma microvesicles (PMVs) and their microRNA content are involved in the development of atherosclerosis and could serve as biomarkers for cardiovascular disease (CVD) progression. However, little is known on how smoking influences the levels of PMVs and microRNA signatures in vivo. Therefore, we aimed to investigate the effects of smoking on circulating PMV levels and CVD-related PMV-derived microRNAs in young, healthy smokers. Twenty young (10 female, 10 male; 25 ± 4 years) healthy smokers (16 ± 6 cigarettes per day for 8 ± 4 years) and age- and sex-matched controls were included in this study. While complete blood count revealed no differences between both groups, smoking significantly enhanced intracellular reactive oxygen species in platelets and leukocytes as well as platelet-leukocyte aggregate formation. Total circulating PMV counts were significantly reduced in smokers, which could be attributed to decreased platelet-derived PMVs. While the number of endothelial PMVs remained unaffected, smoking propagated circulating leukocyte-derived PMVs. Despite reduced total PMVs, PMV-derived microRNA-profiling of six smoker/control pairs revealed a decrease of only a single microRNA, the major platelet-derived microRNA miR-223. Conversely, miR-29b, a microRNA associated with aortic aneurysm and fibrosis, and RNU6-2, a commonly used reference-RNA, were significantly up-regulated. Smoking leads to alterations in the circulating PMV profile and changes in the PMV-derived microRNA signature already in young, healthy adults. These changes may contribute to the development of smoking-related cardiovascular pathologies. Moreover, these smoking-related changes have to be considered when microRNA or PMV profiles are used as disease-specific biomarkers. PMID:24573468

  10. A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function.

    PubMed

    Jiang, Pingping; Wang, Meng; Xue, Ling; Xiao, Yun; Yu, Jialing; Wang, Hui; Yao, Juan; Liu, Hao; Peng, Yanyan; Liu, Hanqing; Li, Haiying; Chen, Ye; Guan, Min-Xin

    2016-07-15

    In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNA(Ala) 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNA(Ala) function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNA(Ala) precursor 5' end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρ(o)) cells, we showed a 41% reduction in the steady-state level of tRNA(Ala) in mutant cybrids. The mutation caused an improperly aminoacylated tRNA(Ala), as suggested by aberrantly aminoacylated tRNA(Ala) and slower electrophoretic mobility of mutated tRNA. A failure in tRNA(Ala) metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNA(Ala) 5655A → G mutation with hypertension. PMID:27161322

  11. Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization

    PubMed Central

    Larsen, Aaron T.; Fahrenbach, Albert C.; Sheng, Jia; Pian, Julia; Szostak, Jack W.

    2015-01-01

    Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s2U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s2U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s2U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s2U:A and s2U:U pairs and their native counterparts. These results indicate that s2U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s2U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s2U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s2U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using 1H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying. PMID:26240387

  12. U2AF1 Mutations Alter Sequence Specificity of pre-mRNA Binding and Splicing

    PubMed Central

    Okeyo-Owuor, Theresa; White, Brian S.; Chatrikhi, Rakesh; Mohan, Dipika R.; Kim, Sanghyun; Griffith, Malachi; Ding, Li; Ketkar-Kulkarni, Shamika; Hundal, Jasreet; Laird, Kholiswa M.; Kielkopf, Clara L.; Ley, Timothy J.; Walter, Matthew J.; Graubert, Timothy A.

    2014-01-01

    We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of patients with de novo myelodysplastic syndromes (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis. PMID:25311244

  13. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects

    SciTech Connect

    Takahashi, Kei; Yokota, Shin-ichi; Tatsumi, Naoyuki; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki

    2013-10-01

    Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two-thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases. - Highlights: • Plasma miRNA profiles were unambiguously different between smokers and non-smokers. • Smoking status might interfere with the diagnosis of disease using plasma miRNAs. • Changes of plasma miRNA profiles may be a signal of smoking-related diseases.

  14. Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae.

    PubMed

    Kaplan, Craig D

    2013-01-01

    Transcription by RNA polymerase II (Pol II), and all RNA polymerases for that matter, may be understood as comprising two cycles. The first cycle relates to the basic mechanism of the transcription process wherein Pol II must select the appropriate nucleoside triphosphate (NTP) substrate complementary to the DNA template, catalyze phosphodiester bond formation, and translocate to the next position on the DNA template. Performing this cycle in an iterative fashion allows the synthesis of RNA chains that can be over one million nucleotides in length in some larger eukaryotes. Overlaid upon this enzymatic cycle, transcription may be divided into another cycle of three phases: initiation, elongation, and termination. Each of these phases has a large number of associated transcription factors that function to promote or regulate the gene expression process. Complicating matters, each phase of the latter transcription cycle are coincident with cotranscriptional RNA processing events. Additionally, transcription takes place within a highly dynamic and regulated chromatin environment. This chromatin environment is radically impacted by active transcription and associated chromatin modifications and remodeling, while also functioning as a major platform for Pol II regulation. This review will focus on our basic knowledge of the Pol II transcription mechanism, and how altered Pol II activity impacts gene expression in vivo in the model eukaryote Saccharomyces cerevisiae. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation. PMID:23022618

  15. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.

    PubMed

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-11-01

    Blood-brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0 · 05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. PMID:24801999

  16. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells

    PubMed Central

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-01-01

    Blood–brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0·05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. PMID:24801999

  17. 3'-modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium.

    PubMed Central

    Hinrichsen, R D; Fraga, D; Reed, M W

    1992-01-01

    The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology. PMID:1528867

  18. Bisphenol A Exposure Leads to Specific MicroRNA Alterations in Placental Cells

    PubMed Central

    Avissar-Whiting, Michele; Veiga, Keila; Uhl, Kristen; Maccani, Matthew; Gagne, Luc; Moen, Erika; Marsit, Carmen J.

    2010-01-01

    Exposure to bisphenol-A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity. PMID:20417706

  19. Basic Mechanisms of RNA Polymerase II Activity and Alteration of Gene Expression in Saccharomyces cerevisiae

    PubMed Central

    Kaplan, Craig D.

    2014-01-01

    Transcription by RNA Polymerase II (Pol II), and all RNA polymerases for that matter, may be understood as comprising two cycles. The first cycle relates to the basic mechanism of the transcription process wherein Pol II must select the appropriate nucleoside triphosphate (NTP) substrate complementary to the DNA template, catalyze phosphodiester bond formation, and translocate to the next position on the DNA template. Performing this cycle in an iterative fashion allows the synthesis of RNA chains that can be over one million nucleotides in length in some larger eukaryotes. Overlaid upon this enzymatic cycle, transcription may be divided into another cycle of three phases: initiation, elongation, and termination. Each of these phases has a large number of associated transcription factors that function to promote or regulate the gene expression process. Complicating matters, each phase of the latter transcription cycle are coincident with cotranscriptional RNA processing events. Additionally, transcription takes place within a highly dynamic and regulated chromatin environment. This chromatin environment is radically impacted by active transcription and associated chromatin modifications and remodeling, while also functioning as a major platform for Pol II regulation. This review will focus on our basic knowledge of the Pol II transcription mechanism, and how altered Pol II activity impacts gene expression in vivo in the model eukaryote Saccharomyces cerevisiae. PMID:23022618

  20. Spaceflight alters expression of microRNA during T-cell activation.

    PubMed

    Hughes-Fulford, Millie; Chang, Tammy T; Martinez, Emily M; Li, Chai-Fei

    2015-12-01

    Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21. PMID:26276131

  1. Functional Alteration of Tumor-infiltrating Myeloid Cells in RNA Adjuvant Therapy.

    PubMed

    Seya, Tsukasa; Shime, Hiroaki; Matsumoto, Misako

    2015-08-01

    Macrophages, as well as dendritic cells (DCs), are derived from myeloid progenitor cells. Recent evidence suggests that tumor-infiltrating macrophages differ in many aspects from conventional tissue macrophages, including nature, function and markers. Tumors usually contain various myeloid lineage cells in their non-parenchymal environment. In immunotherapy for cancer, tumor cells and non-parenchymal cells are exposed to tumor-associated antigens (TAA) and tumor-cell-derived nucleic acids. In addition, a dsRNA mimic, polyinosinic:polycytidylic acid (polyI:C), exhibits strong adjuvant activity, which acts both on the immune system and tumor constituents. Herein we discuss the RNA recognition system and unique cellular output in tumor-associated myeloid cells in response to immunotherapy. We especially focus on the mechanism by which RNA adjuvant alters the tumor-supportive nature of tumor-infiltrated myeloid cells to those with tumoricidal activity. We discuss how RNA administration makes tumor cells collapse and its significance of evoking cell death signals in tumor cells and macrophages. This knowledge will be applicable to the development of an alternative immunotherapy for cancer. PMID:26168476

  2. Niacin in Pharmacological Doses Alters MicroRNA Expression in Skeletal Muscle of Obese Zucker Rats

    PubMed Central

    Most, Erika; Ringseis, Robert; Eder, Klaus

    2014-01-01

    Administration of pharmacological niacin doses was recently reported to have pronounced effects on skeletal muscle gene expression and phenotype in obese Zucker rats, with the molecular mechanisms underlying the alteration of gene expression being completely unknown. Since miRNAs have been shown to play a critical role for gene expression through inducing miRNA-mRNA interactions which results in the degradation of specific mRNAs or the repression of protein translation, we herein aimed to investigate the influence of niacin at pharmacological doses on the miRNA expression profile in skeletal muscle of obese Zucker rats fed either a control diet with 30 mg supplemented niacin/kg diet or a high-niacin diet with 780 mg supplemented niacin/kg diet for 4 wk. miRNA microarray analysis revealed that 42 out of a total of 259 miRNAs were differentially expressed (adjusted P-value <0.05), 20 being down-regulated and 22 being up-regulated, between the niacin group and the control group. Using a biostatistics approach, we could demonstrate that the most strongly up-regulated (log2 ratio ≥0.5) and down-regulated (log2 ratio ≤−0.5) miRNAs target approximately 1,800 mRNAs. Gene-term enrichment analysis showed that many of the predicted target mRNAs from the most strongly regulated miRNAs were involved in molecular processes dealing with gene transcription such as DNA binding, transcription regulator activity, transcription factor binding and in important regulatory pathways such as Wnt signaling and MAPK signaling. In conclusion, the present study shows for the first time that pharmacological niacin doses alter the expression of miRNAs in skeletal muscle of obese Zucker rats and that the niacin-regulated miRNAs target a large set of genes and pathways which are involved in gene regulatory activity indicating that at least some of the recently reported effects of niacin on skeletal muscle gene expression and phenotype in obese Zucker rats are mediated through miRNA-mRNA

  3. Atrial fibrillation alters the microRNA expression profiles of the left atria of patients with mitral stenosis

    PubMed Central

    2014-01-01

    Background Structural changes of the left and right atria associated with atrial fibrillation (AF) in mitral stenosis (MS) patients are well known, and alterations in microRNA (miRNA) expression profiles of the right atria have also been investigated. However, miRNA changes in the left atria still require delineation. This study evaluated alterations in miRNA expression profiles of left atrial tissues from MS patients with AF relative to those with normal sinus rhythm (NSR). Methods Sample tissues from left atrial appendages were obtained from 12 MS patients (6 with AF) during mitral valve replacement surgery. From these tissues, miRNA expression profiles were created and analyzed using a human miRNA microarray. Results were validated via reverse-transcription and quantitative PCR for 5 selected miRNAs. Potential miRNA targets were predicted and their functions and potential pathways analyzed via the miRFocus database. Results The expression levels of 22 miRNAs differed between the AF and NSR groups. Relative to NSR patients, in those with AF the expression levels of 45% (10/22) of these miRNAs were significantly higher, while those of the balance (55%, 12/22) were significantly lower. Potential miRNA targets and molecular pathways were identified. Conclusions AF alters the miRNA expression profiles of the left atria of MS patients. These findings may be useful for the biological understanding of AF in MS patients. PMID:24461008

  4. Altered microRNA expression in bovine skeletal muscle with age.

    PubMed

    Sun, J; Sonstegard, T S; Li, C; Huang, Y; Li, Z; Lan, X; Zhang, C; Lei, C; Zhao, X; Chen, H

    2015-06-01

    Age-dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next-generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty-six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR-1, miR-206 and let-7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up-regulated and 22 miRNAs were down-regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta-mir-10020. A dual luciferase reporter assay was used to demonstrate that bta-mir-10020 directly targeted the 3'-UTR of the bovine ANGPT1 gene. The overexpression of bta-mir-10020 significantly decreased the DsRed fluorescence in the wild-type expression cassette compared to the mutant type. Using three computational approaches - miranda, pita and rnahybrid - these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type. PMID:25703017

  5. Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control.

    PubMed

    Khan, Subuhi; Mackay, John; Liefting, Lia; Ward, Lisa

    2015-09-01

    Apple scar skin viroid (ASSVd) is an important quarantine pathogen for international movement of pome germplasm as it can cause significant damage to pip fruit. A one-step real-time RT-PCR assay was developed for the rapid and sensitive detection of ASSVd. The assay was able to detect a wide range of ASSVd isolates and was highly specific compared to a published conventional RT-PCR. The detection limit of the new assay was estimated to be about 100 copies of the ASSVd target. The assay can be run as a duplex with the nad5 internal control primers and probe to simultaneously check the PCR competency of the samples therefore reducing the risk of false negatives. It is expected that this real-time RT-PCR assay will facilitate efficient testing for ASSVd by regulatory services, and will also have a wider use for the general detection of ASSVd in a range of pip fruit. PMID:25962536

  6. Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

    PubMed Central

    Shaaban, S A; Krupp, B M; Hall, B D

    1995-01-01

    In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes. PMID:7862140

  7. Altered expression and editing of miRNA-100 regulates iTreg differentiation

    PubMed Central

    Negi, Vinny; Paul, Deepanjan; Das, Sudipta; Bajpai, Prashant; Singh, Suchita; Mukhopadhyay, Arijit; Agrawal, Anurag; Ghosh, Balaram

    2015-01-01

    RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4+ T cell differentiation. CD4+ T cells are an integral component of the adaptive immune system. Naïve CD4+ T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end. PMID:26209130

  8. 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.

  9. BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

    PubMed

    Borck, Guntram; Hög, Friederike; Dentici, Maria Lisa; Tan, Perciliz L; Sowada, Nadine; Medeira, Ana; Gueneau, Lucie; Thiele, Holger; Kousi, Maria; Lepri, Francesca; Wenzeck, Larissa; Blumenthal, Ian; Radicioni, Antonio; Schwarzenberg, Tito Livio; Mandriani, Barbara; Fischetto, Rita; Morris-Rosendahl, Deborah J; Altmüller, Janine; Reymond, Alexandre; Nürnberg, Peter; Merla, Giuseppe; Dallapiccola, Bruno; Katsanis, Nicholas; Cramer, Patrick; Kubisch, Christian

    2015-02-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development. PMID:25561519

  10. BRF1 mutations alter RNA polymerase III–dependent transcription and cause neurodevelopmental anomalies

    PubMed Central

    Hög, Friederike; Dentici, Maria Lisa; Tan, Perciliz L.; Sowada, Nadine; Medeira, Ana; Gueneau, Lucie; Thiele, Holger; Kousi, Maria; Lepri, Francesca; Wenzeck, Larissa; Blumenthal, Ian; Radicioni, Antonio; Schwarzenberg, Tito Livio; Mandriani, Barbara; Fischetto, Rita; Morris-Rosendahl, Deborah J.; Altmüller, Janine; Reymond, Alexandre; Nürnberg, Peter; Merla, Giuseppe; Dallapiccola, Bruno; Katsanis, Nicholas; Cramer, Patrick; Kubisch, Christian

    2015-01-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III–related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development. PMID:25561519

  11. Altered expression and editing of miRNA-100 regulates iTreg differentiation.

    PubMed

    Negi, Vinny; Paul, Deepanjan; Das, Sudipta; Bajpai, Prashant; Singh, Suchita; Mukhopadhyay, Arijit; Agrawal, Anurag; Ghosh, Balaram

    2015-09-18

    RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+) T cells are an integral component of the adaptive immune system. Naïve CD4(+) T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end. PMID:26209130

  12. MicroRNA expression is altered in lateral septum across reproductive stages.

    PubMed

    Saul, M C; Zhao, C; Driessen, T M; Eisinger, B E; Gammie, S C

    2016-01-15

    MicroRNAs (miRNAs) inhibit RNA targets and may contribute to postpartum central nervous system (CNS) gene expression changes, although this has never been tested. In the present study, we directly evaluated miRNA levels using RNA sequencing during reproduction in female mice in the lateral septum (LS). We found the reliable and robust changes of miRNAs away from the virgin stage at the three other stages, namely pregnant, day 1 postpartum, and day 8 postpartum. For a given miRNA that was significantly different from the virgin condition in more than one group, the direction of change was always the same. Overall, we identified 32 upregulated miRNAs and 25 downregulated miRNAs that were consistently different from the virgin state. 'Arm switching' occurs for miR-433-3 and miR-7b. Unexpectedly, a third of upregulated miRNAs (relative to virgin) were highly localized within the 12qF1 region of chromosome 12 that includes the Dlk1-Dio3 gene cluster implicated in stem cell and neuronal differentiation. Over 1500 genes were targeted by multiple upregulated miRNAs with about 100 genes targeted by five or more miRNAs. Over 1000 genes were targeted by multiple downregulated miRNAs with about 50 genes targeted by five or more miRNAs. Half of the target genes were regulated by up and downregulated miRNAs, indicating homeostatic regulation. Transcriptional regulation was the most enriched pathway for genes linked to up or down regulated miRNAs. Other enriched pathways included protein kinase activity (e.g., MAP kinase), CNS development, axon guidance, neurotrophin signaling, neuron development/differentiation, and neurogenesis. Previously published postpartum LS gene expression changes were enrichment for LS miRNA targets, as expected. Surprisingly, postpartum gene expression changes from other regions were also enriched against LS miRNA targets, suggesting a core group of miRNAs may act across the CNS during reproduction. Together, we directly examine miRNAs and find

  13. A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure.

    PubMed

    Wang, Ting; Zhou, Tong; Saadat, Laleh; Garcia, Joe G N

    2015-06-01

    Myosin light-chain kinase (MYLK) is a gene known to be significantly associated with severe asthma in African Americans. Here we further examine the molecular function of a single-nucleotide polymorphism (SNP), located in the non-muscle myosin light-chain kinase isoform (nmMLCK), in asthma susceptibility and pathobiology. We identified nmMLCK variant (reference SNP: rs9840993, NM_053025: 721C>T, c.439C>T) with a distinct mRNA secondary structure from the other variants. The nmMLCK variant (721C) secondary structure exhibits increased stability with an elongated half-life in the human endothelial cell, and greater efficiency in protein translation initiation owing to an increased accessibility to translation start site. Finally, nmMLCK expression of 721C- and 721T-containing MYLK transgenes were compared in nmMLCK(-/-) mice and confirmed deleterious effects of nmMLCK expression on asthmatic indices and implicated the augmented influence of MYLK 721C>T (c.439C>T) SNP on asthma severity. The confirmation of the novel mechanism of the regulation of asthmatic inflammation by a MYLK advances knowledge of the genetic basis for asthma disparities, and further suggests the potential of nmMLCK as a therapeutic target. Our study suggests that in addition to altering protein structure and function, non-synonymous SNPs may also lead to phenotypic disparity by altering protein expression. PMID:25271083

  14. Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion

    PubMed Central

    Loupe, J M; Miller, P J; Bonner, B P; Maggi, E C; Vijayaraghavan, J; Crabtree, J S; Taylor, C M; Zabaleta, J; Hollenbach, A D

    2016-01-01

    Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression. PMID:27454080

  15. Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion.

    PubMed

    Loupe, J M; Miller, P J; Bonner, B P; Maggi, E C; Vijayaraghavan, J; Crabtree, J S; Taylor, C M; Zabaleta, J; Hollenbach, A D

    2016-01-01

    Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression. PMID:27454080

  16. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

    PubMed

    O'Neill, J P; Rogan, P K; Cariello, N; Nicklas, J A

    1998-11-01

    The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are

  17. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

  18. Peripheral whole blood microRNA alterations in major depression and bipolar disorder.

    PubMed

    Maffioletti, Elisabetta; Cattaneo, Annamaria; Rosso, Gianluca; Maina, Giuseppe; Maj, Carlo; Gennarelli, Massimo; Tardito, Daniela; Bocchio-Chiavetto, Luisella

    2016-08-01

    Major depression (MD) and bipolar disorder (BD) are severe and potentially life-threating mood disorders whose etiology is to date not completely understood. MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein synthesis post-transcriptionally by base-pairing to target gene mRNAs. Growing evidence indicated that miRNAs might play a key role in the pathogenesis of neuropsychiatric disorders and in the action of psychotropic drugs. On these bases, in this study we evaluated the expression levels of 1733 mature miRNAs annotated in miRBase v.17, through a microarray technique, in the blood of 20 MD and 20 BD patients and 20 healthy controls, in order to identify putative miRNA signatures associated with mood disorders. We found that 5 miRNAs (hsa-let-7a-5p, hsa-let-7d-5p, hsa-let-7f-5p, hsa-miR-24-3p and hsa-miR-425-3p) were specifically altered in MD patients and 5 (hsa-miR-140-3p, hsa-miR-30d-5p, hsa-miR-330-5p, hsa-miR-378a-5p and hsa-miR-21-3p) in BD patients, whereas 2 miRNAs (hsa-miR-330-3p and hsa-miR-345-5p) were dysregulated in both the diseases. The bioinformatic prediction of the genes targeted by the altered miRNAs revealed the possible involvement of neural pathways relevant for psychiatric disorders. In conclusion, the observed results indicate a dysregulation of miRNA blood expression in mood disorders and could indicate new avenues for a better understanding of their pathogenetic mechanisms. The identified alterations may represent potential peripheral biomarkers to be complemented with other clinical and biological features for the improvement of diagnostic accuracy. PMID:27152760

  19. Intestinal Epithelial Barrier Disruption through Altered Mucosal MicroRNA Expression in Human Immunodeficiency Virus and Simian Immunodeficiency Virus Infections

    PubMed Central

    Gaulke, Christopher A.; Porter, Matthew; Han, Yan-Hong; Sankaran-Walters, Sumathi; Grishina, Irina; George, Michael D.; Dang, Angeline T.; Ding, Shou-Wei; Jiang, Guochun; Korf, Ian

    2014-01-01

    ABSTRACT Epithelial barrier dysfunction during human immunodeficiency virus (HIV) infection has largely been attributed to the rapid and severe depletion of CD4+ T cells in the gastrointestinal (GI) tract. Although it is known that changes in mucosal gene expression contribute to intestinal enteropathy, the role of small noncoding RNAs, specifically microRNA (miRNA), has not been investigated. Using the simian immunodeficiency virus (SIV)-infected nonhuman primate model of HIV pathogenesis, we investigated the effect of viral infection on miRNA expression in intestinal mucosa. SIV infection led to a striking decrease in the expression of mucosal miRNA compared to that in uninfected controls. This decrease coincided with an increase in 5′-3′-exoribonuclease 2 protein and alterations in DICER1 and Argonaute 2 expression. Targets of depleted miRNA belonged to molecular pathways involved in epithelial proliferation, differentiation, and immune response. Decreased expression of several miRNA involved in maintaining epithelial homeostasis in the gut was localized to the proliferative crypt region of the intestinal epithelium. Our findings suggest that SIV-induced decreased expression of miRNA involved in epithelial homeostasis, disrupted expression of miRNA biogenesis machinery, and increased expression of XRN2 are involved in the development of epithelial barrier dysfunction and gastroenteropathy. IMPORTANCE MicroRNA (miRNA) regulate the development and function of intestinal epithelial cells, and many viruses disrupt normal host miRNA expression. In this study, we demonstrate that SIV and HIV disrupt expression of miRNA in the small intestine during infection. The depletion of several key miRNA is localized to the proliferative crypt region of the gut epithelium. These miRNA are known to control expression of genes involved in inflammation, cell death, and epithelial maturation. Our data indicate that this disruption might be caused by altered expression of miRNA

  20. Cocaine treatment alters oxytocin receptor binding but not mRNA production in postpartum rat dams.

    PubMed

    Jarrett, T M; McMurray, M S; Walker, C H; Johns, J M

    2006-06-01

    Gestational cocaine treatment in rat dams results in decreased oxytocin (OT) levels, up-regulated oxytocin receptor (OTR) binding density and decreased receptor affinity in the whole amygdala, all concomitant with a significant increase in maternal aggression on postpartum day six. Rat dams with no gestational drug treatment that received an infusion of an OT antagonist directly into the central nucleus of the amygdala (CeA) exhibited similarly high levels of maternal aggression towards intruders. Additionally, studies indicate that decreased OT release from the hypothalamic division of the paraventricular nucleus (PVN) is coincident with heightened maternal aggression in rats. Thus, it appears that cocaine-induced alterations in OT system dynamics (levels, receptors, production, and/or release) may mediate heightened maternal aggression following cocaine treatment, but the exact mechanisms through which cocaine impacts the OT system have not yet been determined. Based on previous studies, we hypothesized that two likely mechanisms of cocaine's action would be, increased OTR binding specifically in the CeA, and decreased OT mRNA production in the PVN. Autoradiography and in situ hybridization assays were performed on targeted nuclei in brain regions of rat dams on postpartum day six, following gestational treatment twice daily with cocaine (15 mg/kg) or normal saline (1 ml/kg). We now report cocaine-induced reductions in OTR binding density in the ventromedial hypothalamus (VMH) and bed nucleus of the stria terminalis (BNST), but not the CeA. There was no significant change in OT mRNA production in the PVN following cocaine treatment. PMID:16677710

  1. Amphetamine-induced c-fos mRNA expression is altered in rats with neonatal ventral hippocampal damage.

    PubMed

    Lillrank, S M; Lipska, B K; Bachus, S E; Wood, G K; Weinberger, D R

    1996-08-01

    To further characterize the mechanisms underlying enhanced dopamine-related behaviors expressed during adulthood in rats with neonatal excitotoxic ventral hippocampal (VH) damage, we studied the expression of c-fos mRNA in these rats after a single saline or amphetamine (AMPH) (10 mg/kg, i.p.) injection using in situ hybridization. The VH of rat pups was lesioned with ibotenic acid on postnatal day 7 (PD7). At the age of 90 days, rats were challenged with AMPH or saline, and the expression of c-fos mRNA using an oligonucleotide probe was assessed 30, 90, and 180 min later. AMPH significantly increased c-fos mRNA expression in medial prefrontal cortex, piriform cortex, cingulate cortex, septal region, and dorsolateral and ventromedial striatum in control and lesioned rats. However, this response to AMPH was attenuated 30 min after AMPH injection in all of these regions in the lesioned as compared to the sham-operated rats. No significant changes were seen at other time points. These results indicate that the neonatal VH lesion alters time-dependent intracellular signal transduction mechanisms measured by AMPH-induced c-fos mRNA expression in cortical and subcortical brain regions. Changes in c-fos mRNA expression in this putative animal model of schizophrenia may have implications for long-term alterations in cellular phenotype because of altered regulation of certain target genes. PMID:8855514

  2. Prediction of Altered 3′- UTR miRNA-Binding Sites from RNA-Seq Data: The Swine Leukocyte Antigen Complex (SLA) as a Model Region

    PubMed Central

    Endale Ahanda, Marie-Laure; Fritz, Eric R.; Estellé, Jordi; Hu, Zhi-Liang; Madsen, Ole; Groenen, Martien A. M.; Beraldi, Dario; Kapetanovic, Ronan; Hume, David A.; Rowland, Robert R. R.; Lunney, Joan K.; Rogel-Gaillard, Claire; Reecy, James M.; Giuffra, Elisabetta

    2012-01-01

    The SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3′-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation

  3. Human papillomavirus alters microRNA profiles in squamous cell carcinoma of the head and neck (SCCHN) cell lines

    PubMed Central

    Wald, Abigail I.; Hoskins, Elizabeth E.; Wells, Susanne I.; Ferris, Robert L.; Khan, Saleem A.

    2010-01-01

    Background Human papillomavirus (HPV) positive cases of squamous cell carcinoma of the head and neck (SCCHN) have a much better disease outcome compared to SCCHN cases lacking HPVs. Differences in microRNA (miRNA) expression may affect their clinical outcomes. Methods miRNA expression was studied using microarrays and quantitative RT-PCR in HPV-16 positive and HPV-negative SCCHN cell lines. The role of HPV-16 E6 and E7 oncogenes in altering miRNA expression was investigated using human foreskin keratinocytes (HFKs). Results MiRNAs miR-363, miR-33 and miR-497 were upregulated while miR-155, miR-181a, miR-181b, miR-29a, miR-218, miR-222, miR-221 and miR-142-5p were downregulated in HPV-positive cells compared to both HPV-negative SCCHN and normal oral keratinocytes. HPV-16 E6 oncogene altered miRNA expression in HFKs and in an HPV-16 positive cell line with E6 knockdown using siRNA. Conclusions MiRNAs differentially expressed in the presence of HPV-16 may provide biomarkers for SCCHN and identify cellular pathways targeted by this virus. PMID:20652977

  4. Double-Stranded RNA Resists Condensation

    NASA Astrophysics Data System (ADS)

    Li, Li; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois

    2011-03-01

    Much attention has been focused on DNA condensation because of its fundamental biological importance. The recent discovery of new roles for RNA duplexes demands efficient packaging of double-stranded RNA for therapeutics. Here we report measurements of short DNA and RNA duplexes in the presence of trivalent ions. Under conditions where UV spectroscopy indicates condensation of DNA duplexes into (insoluble) precipitates, RNA duplexes remain soluble. Small angle x-ray scattering results suggest that the differing surface topologies of RNA and DNA may be crucial in generating the attractive forces that result in precipitation.

  5. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  6. RNA-Seq identifies key reproductive gene expression alterations in response to cadmium exposure.

    PubMed

    Hu, Hanyang; Lu, Xing; Cen, Xiang; Chen, Xiaohua; Li, Feng; Zhong, Shan

    2014-01-01

    Cadmium is a common toxicant that is detrimental to many tissues. Although a number of transcriptional signatures have been revealed in different tissues after cadmium treatment, the genes involved in the cadmium caused male reproductive toxicity, and the underlying molecular mechanism remains unclear. Here we observed that the mice treated with different amount of cadmium in their rodent chow for six months exhibited reduced serum testosterone. We then performed RNA-seq to comprehensively investigate the mice testicular transcriptome to further elucidate the mechanism. Our results showed that hundreds of genes expression altered significantly in response to cadmium treatment. In particular, we found several transcriptional signatures closely related to the biological processes of regulation of hormone, gamete generation, and sexual reproduction, respectively. The expression of several testosterone synthetic key enzyme genes, such as Star, Cyp11a1, and Cyp17a1, were inhibited by the cadmium exposure. For better understanding of the cadmium-mediated transcriptional regulatory mechanism of the genes, we computationally analyzed the transcription factors binding sites and the mircoRNAs targets of the differentially expressed genes. Our findings suggest that the reproductive toxicity by cadmium exposure is implicated in multiple layers of deregulation of several biological processes and transcriptional regulation in mice. PMID:24982889

  7. CBR antimicrobials alter coupling between the bridge helix and the β subunit in RNA polymerase

    PubMed Central

    Malinen, Anssi M.; NandyMazumdar, Monali; Turtola, Matti; Malmi, Henri; Grocholski, Thadee; Artsimovitch, Irina; Belogurov, Georgiy A

    2014-01-01

    Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit. PMID:24598909

  8. RNA-Seq Identifies Key Reproductive Gene Expression Alterations in Response to Cadmium Exposure

    PubMed Central

    Hu, Hanyang; Lu, Xing; Cen, Xiang; Chen, Xiaohua; Li, Feng; Zhong, Shan

    2014-01-01

    Cadmium is a common toxicant that is detrimental to many tissues. Although a number of transcriptional signatures have been revealed in different tissues after cadmium treatment, the genes involved in the cadmium caused male reproductive toxicity, and the underlying molecular mechanism remains unclear. Here we observed that the mice treated with different amount of cadmium in their rodent chow for six months exhibited reduced serum testosterone. We then performed RNA-seq to comprehensively investigate the mice testicular transcriptome to further elucidate the mechanism. Our results showed that hundreds of genes expression altered significantly in response to cadmium treatment. In particular, we found several transcriptional signatures closely related to the biological processes of regulation of hormone, gamete generation, and sexual reproduction, respectively. The expression of several testosterone synthetic key enzyme genes, such as Star, Cyp11a1, and Cyp17a1, were inhibited by the cadmium exposure. For better understanding of the cadmium-mediated transcriptional regulatory mechanism of the genes, we computationally analyzed the transcription factors binding sites and the mircoRNAs targets of the differentially expressed genes. Our findings suggest that the reproductive toxicity by cadmium exposure is implicated in multiple layers of deregulation of several biological processes and transcriptional regulation in mice. PMID:24982889

  9. Characterization of a novel RNA polymerase mutant that alters DksA activity.

    PubMed

    Satory, Dominik; Halliday, Jennifer A; Sivaramakrishnan, Priya; Lua, Rhonald C; Herman, Christophe

    2013-09-01

    The auxiliary factor DksA is a global transcription regulator and, with the help of ppGpp, controls the nutritional stress response in Escherichia coli. Although the consequences of its modulation of RNA polymerase (RNAP) are becoming better explained, it is still not fully understood how the two proteins interact. We employed a series of genetic suppressor selections to find residues in RNAP that alter its sensitivity to DksA. Our approach allowed us to identify and genetically characterize in vivo three single amino acid substitutions: β' E677G, β V146F, and β G534D. We demonstrate that the mutation β' E677G affects the activity of both DksA and its homolog, TraR, but does not affect the action of other secondary interactors, such as GreA or GreB. Our mutants provide insight into how different auxiliary transcription factors interact with RNAP and contribute to our understanding of how different stages of transcription are regulated through the secondary channel of RNAP in vivo. PMID:23852871

  10. Considerations in Duplex Investment.

    ERIC Educational Resources Information Center

    Wright, Arthur; Goen, Tom

    Problems of duplex investment are noted in the introduction to this booklet designed to provide a technique by which the investment decision can be approached, develop estimates of typical costs and returns under differing conditions, and encourage investors to analyze objectives and conditions before the decision to buy or build is made. A…

  11. Duplex tab exhaust nozzle

    NASA Technical Reports Server (NTRS)

    Gutmark, Ephraim Jeff (Inventor); Martens, Steven (nmn) (Inventor)

    2012-01-01

    An exhaust nozzle includes a conical duct terminating in an annular outlet. A row of vortex generating duplex tabs are mounted in the outlet. The tabs have compound radial and circumferential aft inclination inside the outlet for generating streamwise vortices for attenuating exhaust noise while reducing performance loss.

  12. The Duplex Society.

    ERIC Educational Resources Information Center

    Schorr, Alvin L.

    1984-01-01

    The duplex society, in which the poor live in close proximity to others but in a separate compartment, is already with us. Unless something deeply changes about family income, more than one-third of future generations will come to adulthood having spent a portion of their childhood in official poverty. (RM)

  13. Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

    PubMed Central

    Casara, Silvia; Sales, Gabriele; Lanfranchi, Gerolamo; Celotti, Lucia; Mognato, Maddalena

    2012-01-01

    Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. PMID:22347458

  14. Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket".

    PubMed

    McClain, W H; Foss, K

    1988-09-30

    The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine. PMID:2459773

  15. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  16. YB-1 and MTA1 protein levels and not DNA or mRNA alterations predict for prostate cancer recurrence

    PubMed Central

    Sheridan, Christine Moore; Grogan, Tristan R.; Nguyen, Hao G.; Galet, Colette; Rettig, Matthew B.; Hsieh, Andrew C.; Ruggero, Davide

    2015-01-01

    Attempts to identify biomarkers to detect prostate tumorigenesis, and thus minimize prostate cancer progression and inform treatment decisions have primarily focused on alterations at the DNA and mRNA levels, ignoring alterations at the level of protein synthesis control. We have previously shown that the PI3K-AKT-mTOR pathway, frequently deregulated in prostate cancer, specifically induces the synthesis of proteins that contribute to metastasis, most notably YB-1 and MTA1, without altering mRNA levels thereby demonstrating the importance of translation control in driving the expression of these genes in cancer. Here, we analyze genomic sequencing and mRNA expression databases, as well as protein expression employing an annotated tissue microarray generated from 332 prostate cancer patients with 15 years of clinical follow-up to determine the combined prognostic capability of YB-1 and MTA1 alterations in forecasting prostate cancer outcomes. Remarkably, protein abundance, but not genomic or transcriptional alterations of YB-1 and MTA1, is predictive of disease recurrence, exhibiting a dose-dependent effect on time to PSA recurrence, an indicator of tumor relapse. Moreover, high protein levels of YB-1 and MTA1 are associated with a 3-fold increased risk for requiring future hormone therapy or radiation therapy. Importantly, YB-1 and MTA1 protein levels significantly increase the predictive capacity of a clinical model for prostate cancer recurrence. These findings demonstrate that protein abundance of YB-1 and MTA1, irrespective of DNA or mRNA status, can predict for prostate cancer relapse and uncover a vast underappreciated repository of biomarkers regulated at the level of protein expression. PMID:25797255

  17. Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome.

    PubMed

    Sellier, Chantal; Freyermuth, Fernande; Tabet, Ricardos; Tran, Tuan; He, Fang; Ruffenach, Frank; Alunni, Violaine; Moine, Herve; Thibault, Christelle; Page, Adeline; Tassone, Flora; Willemsen, Rob; Disney, Matthew D; Hagerman, Paul J; Todd, Peter K; Charlet-Berguerand, Nicolas

    2013-03-28

    Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery. PMID:23478018

  18. Sequestration of DROSHA and DGCR8 by Expanded CGG RNA Repeats Alters MicroRNA Processing in Fragile X-Associated Tremor/Ataxia Syndrome

    PubMed Central

    Sellier, Chantal; Freyermuth, Fernande; Tabet, Ricardos; Tran, Tuan; He, Fang; Ruffenach, Frank; Alunni, Violaine; Moine, Herve; Thibault, Christelle; Page, Adeline; Tassone, Flora; Willemsen, Rob; Disney, Matthew D.; Hagerman, Paul J.; Todd, Peter K.; Charlet-Berguerand, Nicolas

    2013-01-01

    SUMMARY Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55–200 CGG repeats in the 5′ UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of micro-RNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery. PMID:23478018

  19. A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function

    PubMed Central

    Jiang, Pingping; Wang, Meng; Xue, Ling; Xiao, Yun; Yu, Jialing; Wang, Hui; Yao, Juan; Liu, Hao; Peng, Yanyan; Liu, Hanqing; Li, Haiying; Chen, Ye

    2016-01-01

    In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNAAla 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension. PMID:27161322

  20. A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase.

    PubMed

    Lin, Y H; Yadav, P; Ravatn, R; Stollar, V

    2000-06-20

    Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of virus was reduced 100- to 1000-fold. By serial passage of our standard SV(STD) in Ae. albopictus cells in the presence of increasing concentrations of PZF, a mutant, SV(PZF), was derived, which was not inhibited by PZF. SV(PZF) is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease levels of UTP and CTP. Analysis of chimeric viruses containing sequences from the SV(PZF) and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region identified four mutations (nt 5750, 6627, 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T in nsP4. Characterization of viruses containing one or more of these mutations demonstrated that all three mutations in the nsP4 coding region are required to produce full resistance to PZF. Using a molecular model of nsP4 based on the structure of HIV reverse transcriptase, we located amino acid change M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these three amino acid changes in nsP4 alter the geometry of the NTP binding pocket so as to increase the affinity of the enzyme for CTP and UTP. PMID:10873749

  1. Altered Gene Expression Associated with microRNA Binding Site Polymorphisms

    PubMed Central

    Võsa, Urmo; Esko, Tõnu; Kasela, Silva; Annilo, Tarmo

    2015-01-01

    Allele-specific gene expression associated with genetic variation in regulatory regions can play an important role in the development of complex traits. We hypothesized that polymorphisms in microRNA (miRNA) response elements (MRE-SNPs) that either disrupt a miRNA binding site or create a new miRNA binding site can affect the allele-specific expression of target genes. By integrating public expression quantitative trait locus (eQTL) data, miRNA binding site predictions, small RNA sequencing, and Argonaute crosslinking immunoprecipitation (AGO-CLIP) datasets, we identified genetic variants that can affect gene expression by modulating miRNA binding efficiency. We also identified MRE-SNPs located in regions associated with complex traits, indicating possible causative mechanisms associated with these loci. The results of this study expand the current understanding of gene expression regulation and help to interpret the mechanisms underlying eQTL effects. PMID:26496489

  2. Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart.

    PubMed

    Charlemagne, D; Orlowski, J; Oliviero, P; Rannou, F; Sainte Beuve, C; Swynghedauw, B; Lane, L K

    1994-01-14

    To determine if an altered expression of the Na,K-ATPase alpha isoform genes is responsible for an observed increase in cardiac glycoside sensitivity in compensatory hypertrophy, we performed Northern and slot blot analyses of RNA and specific immunological detection of Na,K-ATPase isoforms in rat hearts from normal and pressure overload-treated animals induced by abdominal aortic constriction. During the early phase of hypertrophy, the only alteration is a decrease in the alpha 2 mRNA isoform. In the compensated hypertrophied heart, the levels of the predominant alpha 1 isoform (mRNA and protein) and the beta 1 subunit mRNA are unchanged. In contrast, the alpha 2 isoform (mRNA and protein) is decreased by 35% and up to 61-64% in mild (< 55%) and severe (> 55%) hypertrophy, respectively. The alpha 3 isoform (mRNA and protein), which is extremely low in adult heart, is increased up to 2-fold during hypertrophy but accounts for only approximately equal to 5% of the total alpha isoform mRNA. These findings demonstrate that, in cardiac hypertrophy, the three alpha isoforms of the Na,K-ATPase are independently regulated and that regulation occurs at a pretranslational level. The pattern of expression in hypertrophied adult heart is similar to that of the neonatal heart where the inverse regulation between the alpha 2 and alpha 3 ouabain high affinity isoforms has been reported. This suggests that distinct regulatory mechanisms controlling Na,K-ATPase isoform expression may, at least in part, be involved in the sensitivity to cardiac glycosides. PMID:8288620

  3. Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions.

    PubMed

    Berman, Y; Devi, L; Spangler, R; Kreek, M J; Carr, K D

    1997-06-01

    It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need. PMID:9191075

  4. Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors

    PubMed Central

    Watanabe, Yoshihisa; Yoshimoto, Kanji; Tatebe, Harutsugu; Kita, Masakazu; Nishikura, Kazuko; Kimura, Minoru; Tanaka, Masaki

    2014-01-01

    Serotonin 2C receptors (5-HT2CR) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT2CR mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT2CR mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT2CR, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT2CR mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT2CR mRNA editing underlie alcohol preference in mice. PMID:24345557

  5. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations.

    PubMed

    Martin, Dorrelyn P; Miya, Jharna; Reeser, Julie W; Roychowdhury, Sameek

    2016-01-01

    RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations. PMID:27585245

  6. Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

    PubMed Central

    Somasundaram, Kumaravel

    2015-01-01

    Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM- EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes- ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines. PMID:26496030

  7. Neuroprotection induced by post-conditioning following ischemia/reperfusion in mice is associated with altered microRNA expression.

    PubMed

    Miao, Wei; Bao, Tian-Hao; Han, Jian-Hong; Yin, Mei; Zhang, Jie; Yan, Yong; Zhu, Yu-Hong

    2016-09-01

    Ischemic preconditioning and ischemic postconditioning (IPostC) represent promising strategies to reduce ischemia-reperfusion (I/R) injury and attenuate the lethal ischemic damage following stroke. However, the mechanism underlying this attenuation remains to be elucidated. It was hypothesized that alterations in microRNA (miRNA) expression in the cerebral cortex and hippocampus of mice following I/R is associated with the functional improvement induced by IPostC. Behavioral changes were assessed in a mouse model of I/R in the absence or presence of IPostC, followed by microarray analyses to investigate the expressional alterations of miRNAs in the cerebral cortex and hippocampus of mice. The results of the present study revealed that IPostC abrogated the neurological impairment and hippocampus‑associated cognitive deficits induced by I/R, and upregulated or downregulated the expression levels of numerous miRNAs. Furthermore, the upregulation of miR‑19a, and the downregulation of miR‑1, let‑7f and miR‑124 expression levels following IPostC was confirmed utilizing reverse transcription‑quantitative polymerase chain reaction. The results of the present study demonstrated that alterations in miRNA expression in the cerebral cortex and hippocampus of mice following I/R was associated with the neuroprotection induced by IPostC. PMID:27485299

  8. Neuroprotection induced by post-conditioning following ischemia/reperfusion in mice is associated with altered microRNA expression

    PubMed Central

    Miao, Wei; Bao, Tian-Hao; Han, Jian-Hong; Yin, Mei; Zhang, Jie; Yan, Yong; Zhu, Yu-Hong

    2016-01-01

    Ischemic preconditioning and ischemic postconditioning (IPostC) represent promising strategies to reduce ischemia-reperfusion (I/R) injury and attenuate the lethal ischemic damage following stroke. However, the mechanism underlying this attenuation remains to be elucidated. It was hypothesized that alterations in microRNA (miRNA) expression in the cerebral cortex and hippocampus of mice following I/R is associated with the functional improvement induced by IPostC. Behavioral changes were assessed in a mouse model of I/R in the absence or presence of IPostC, followed by microarray analyses to investigate the expressional alterations of miRNAs in the cerebral cortex and hippocampus of mice. The results of the present study revealed that IPostC abrogated the neurological impairment and hippocampus-associated cognitive deficits induced by I/R, and upregulated or downregulated the expression levels of numerous miRNAs. Furthermore, the upregulation of miR-19a, and the downregulation of miR-1, let-7f and miR-124 expression levels following IPostC was confirmed utilizing reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that alterations in miRNA expression in the cerebral cortex and hippocampus of mice following I/R was associated with the neuroprotection induced by IPostC. PMID:27485299

  9. Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

    PubMed

    Hashikawa, Naoya; Ogawa, Takumi; Sakamoto, Yusuke; Ogawa, Mami; Matsuo, Yumi; Zamami, Yoshito; Hashikawa-Hobara, Narumi

    2015-08-01

    Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test. PMID:25820756

  10. MicroRNA abundance is altered in synaptoneurosomes during prion disease.

    PubMed

    Boese, Amrit S; Saba, Reuben; Campbell, Kristyn; Majer, Anna; Medina, Sarah; Burton, Lynn; Booth, Timothy F; Chong, Patrick; Westmacott, Garrett; Dutta, Sucharita M; Saba, Julian A; Booth, Stephanie A

    2016-03-01

    Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p). PMID:26658803

  11. Altered Expression of Circulating MicroRNA in Plasma of Patients with Primary Osteoarthritis and In Silico Analysis of Their Pathways

    PubMed Central

    Borgonio Cuadra, Verónica M.; González-Huerta, Norma Celia; Romero-Córdoba, Sandra; Hidalgo-Miranda, Alfredo; Miranda-Duarte, Antonio

    2014-01-01

    Objective To analyze a set of circulating microRNA (miRNA) in plasma from patients with primary Osteoarthritis (OA) and describe the biological significance of altered miRNA in OA based on an in silico analysis of their target genes. Methods miRNA expression was analyzed using TaqMan Low Density Arrays and independent assays. The search for potential messenger RNA (mRNA) targets of the differentially expressed miRNA was performed by means of the miRWalk and miRecords database; we conducted the biological relevance of the predicted miRNA targets by pathway analysis with the Reactome and DAVID databases. Results We measured the expression of 380 miRNA in OA; 12 miRNA were overexpressed under the OA condition (p value, ≤0.05; fold change, >2). These results were validated by the detection of some selected miRNA by quantitative PCR (qPCR). In silico analysis showed that target messenger RNA (mRNA) were potentially regulated by these miRNA, including genes such as SMAD1, IL-1B, COL3A, VEGFA, and FGFR1, important in chondrocyte maintenance and differentiation. Some metabolic pathways affected by the miRNA: mRNA ratio are signaling Bone morphogenetic proteins (BMP), Platelet-derived growth factor (PDGF), and Nerve growth factor (NGF), these latter two involved in the process of pain. Conclusions We identified 12 miRNA in the plasma of patients with primary OA. Specific miRNA that are altered in the disease could be released into plasma, either due to cartilage damage or to an inherent cellular mechanism. Several miRNA could regulate genes and pathways related with development of the disease; eight of these circulating miRNA are described, to our knowledge, for first time in OA. PMID:24901787

  12. Alterations in SiRNA and MiRNA Expression Profiles Detected by Deep Sequencing of Transgenic Rice with SiRNA-Mediated Viral Resistance

    PubMed Central

    Wang, Xifeng; Liang, Chun

    2015-01-01

    RNA-mediated gene silencing has been demonstrated to serve as a defensive mechanism against viral pathogens by plants. It is known that specifically expressed endogenous siRNAs and miRNAs are involved in the self-defense process during viral infection. However, research has been rarely devoted to the endogenous siRNA and miRNA expression changes under viral infection if the resistance has already been genetically engineered in plants. Aiming to gain a deeper understanding of the RNA-mediated gene silencing defense process in plants, the expression profiles of siRNAs and miRNAs before and after viral infection in both wild type and transgenic anti-Rice stripe virus (RSV) rice plants were examined by small RNA high-throughput sequencing. Our research confirms that the newly generated siRNAs, which are derived from the engineered inverted repeat construct, is the major contributor of the viral resistance in rice. Further analysis suggests the accuracy of siRNA biogenesis might be affected when siRNAs machinery is excessively used in the transgenic plants. In addition, the expression levels of many known miRNAs are dramatically changed due to RSV infection on both wild type and transgenic rice plants, indicating potential function of those miRNAs involved in plant-virus interacting process. PMID:25559820

  13. Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress.

    PubMed

    Jacometo, Carolina B; Osorio, Johan S; Socha, Michael; Corrêa, Marcio N; Piccioli-Cappelli, Fiorenzo; Trevisi, Erminio; Loor, Juan J

    2015-11-01

    Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40 mg/kg of Zn, 20 mg/kg of Mn, 5 mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75 mg/kg of Zn, 65 mg/kg of Mn, 11 mg/kg of Cu, and 1 mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24 h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and

  14. An RNA-seq Protocol to Identify mRNA Expression Changes in Mouse Diaphyseal Bone: Applications in Mice with Bone Property Altering Lrp5 Mutations

    PubMed Central

    Ayturk, Ugur M.; Jacobsen, Christina M.; Christodoulou, Danos C.; Gorham, Joshua; Seidman, Jonathan G.; Seidman, Christine E.; Robling, Alexander G.; Warman, Matthew L.

    2013-01-01

    Loss-of-function and certain missense mutations in the Wnt co-receptor LRP5 significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knockin mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5−/−), and high bone mass (HBM)-causing (Lrp5p.A214V/+) alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating non-skeletal tissue (i.e., blood, bone marrow, and skeletal muscle) in our data. These methods included pre-digestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal’s right and left tibiae and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 “sufficient” (i.e., Lrp5+/+ and Lrp5p.A214V/+) and “insufficient” (Lrp5−/−) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone. PMID:23553928

  15. Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

    PubMed Central

    Oakes, M; Nogi, Y; Clark, M W; Nomura, M

    1993-01-01

    We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants. Images PMID:8455621

  16. Genes and Small RNA Transcripts Exhibit Dosage-Dependent Expression Pattern in Maize Copy-Number Alterations.

    PubMed

    Zuo, Tao; Zhang, Jianbo; Lithio, Andrew; Dash, Sudhansu; Weber, David F; Wise, Roger; Nettleton, Dan; Peterson, Thomas

    2016-07-01

    Copy-number alterations are widespread in animal and plant genomes, but their immediate impact on gene expression is still unclear. In animals, copy-number alterations usually exhibit dosage effects, except for sex chromosomes which tend to be dosage compensated. In plants, genes within small duplications (<100 kb) often exhibit dosage-dependent expression, whereas large duplications (>50 Mb) are more often dosage compensated. However, little or nothing is known about expression in moderately-sized (1-50 Mb) segmental duplications, and about the response of small RNAs to dosage change. Here, we compared maize (Zea mays) plants with two, three, and four doses of a 14.6-Mb segment of chromosome 1 that contains ∼300 genes. Plants containing the duplicated segment exhibit dosage-dependent effects on ear length and flowering time. Transcriptome analyses using GeneChip and RNA-sequencing methods indicate that most expressed genes and unique small RNAs within the duplicated segments exhibit dosage-dependent transcript levels. We conclude that dosage effect is the predominant regulatory response for both genes and unique small RNA transcripts in the segmental dosage series we tested. To our knowledge this is the first analysis of small RNA expression in plant gene dosage variants. Because segmental duplications comprise a significant proportion of eukaryotic genomes, these findings provide important new insight into the regulation of genes and small RNAs in response to dosage changes. PMID:27129738

  17. Global Assessment of Antrodia cinnamomea-Induced MicroRNA Alterations in Hepatocarcinoma Cells

    PubMed Central

    Chan, Yu-Tzu; Huang, Yu-Feng; Ma, Nianhan; Yu, Alice L.; Wu, Chung-Yi; Hu, Miao-Lin; Chiu, Kuo Ping

    2013-01-01

    Recent studies have demonstrated a potent anticancer potential of medicinal fungus Antrodia cinnamomea, especially against hepatocarcinoma. These studies, however, were performed with prolonged treatments, and the early anticancer events remain missing. To probe the early anticancer mechanisms of A. cinnamomea, we treated SK-Hep-1 liver cancer cell with A. cinnamomea fruiting body extract for 2 and 4 hours, sequenced RNA samples with next-generation sequencing approach, and profiled the genome-wide miRNA and mRNA transcriptomes. Results unmistakably associated the early anticancer effect of A. cinnamomea fruiting body extract with a global downregulation of miRNAs which occurred solely in the A. cinnamomea fruiting body extract-treated SK-Hep-1 cells. Moreover, the inhibitory effect of A. cinnamomea fruiting body extract upon cancer miRNAs imposed no discrimination against any particular miRNA species, with oncomirs miR-21, miR-191 and major oncogenic clusters miR-17-92 and miR-106b-25 among the most severely downregulated. Western blotting further indicated a decrease in Drosha and Dicer proteins which play a key role in miRNA biogenesis, together with an increase of XRN2 known to participate in miRNA degradation pathway. Transcriptome profiling followed by GO and pathway analyses indicated that A. cinnamomea induced apoptosis, which was tightly associated with a downregulation of PI3K/AKT and MAPK pathways. Phosphorylation assay further suggested that JNK and c-Jun were closely involved in the apoptotic process. Taken together, our data indicated that the anticancer effect of A. cinnamomea can take place within a few hours by targeting multiple proteins and the miRNA system. A. cinnamomea indiscriminately induced a global downregulation of miRNAs by simultaneously inhibiting the key enzymes involved in miRNA maturation and activating XRN2 protein involved in miRNA degradation. Collapsing of the miRNA system together with downregulation of cell growth and survival

  18. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  19. Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae

    PubMed Central

    2013-01-01

    Background The rice blast fungus, Magnaporthe oryzae is a destructive pathogen of rice and other related crops, causing significant yield losses worldwide. Endogenous small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs) are critical components of gene regulation in many eukaryotic organisms. Recently several new species of sRNAs have been identified in fungi. This fact along with the availability of genome sequence makes M. oryzae a compelling target for sRNA profiling. We have examined sRNA species and their biosynthetic genes in M. oryzae, and the degree to which these elements regulate fungal stress responses. To this end, we have characterized sRNAs under different physiological stress conditions, which had not yet been examined in this fungus. Results The resulting libraries are composed of more than 37 million total genome matched reads mapping to intergenic regions, coding sequences, retrotransposons, inverted, tandem, and other repeated regions of the genome with more than half of the small RNAs arising from intergenic regions. The 24 nucleotide (nt) size class of sRNAs was predominant. A comparison to transcriptional data of M. oryzae undergoing the same physiological stresses indicates that sRNAs play a role in transcriptional regulation for a small subset of genes. Support for this idea comes from generation and characterization of mutants putatively involved in sRNAs biogenesis; our results indicate that the deletion of Dicer-like genes and an RNA-Dependent RNA Polymerase gene increases the transcriptional regulation of this subset of genes, including one involved in virulence. Conclusions Various physiological stressors and in planta conditions alter the small RNA profile of the rice blast fungus. Characterization of sRNA biosynthetic mutants helps to clarify the role of sRNAs in transcriptional control. PMID:23663523

  20. Altered microRNA profiles in cerebrospinal fluid exosome in Parkinson disease and Alzheimer disease

    PubMed Central

    Gui, YaXing; Liu, Hai; Zhang, LiShan; Lv, Wen; Hu, XingYue

    2015-01-01

    The differential diagnosis of Parkinson's diseases (PD) is challenging, especially in the early stages of the disease. We developed a microRNA profiling strategy for exosomal miRNAs isolated from cerebrospinal fluid (CSF) in PD and AD. Sixteen exosomal miRNAs were up regulated and 11 miRNAs were under regulated significantly in PD CSF when compared with those in healthy controls (relative fold > 2, p < 0.05). MiR-1 and miR-19b-3p were validated and significantly reduced in independent samples. While miR-153, miR-409-3p, miR-10a-5p, and let-7g-3p were significantly over expressed in PD CSF exosome. Bioinformatic analysis by DIANA-mirPath demonstrated that Neurotrophin signaling, mTOR signaling, Ubiquitin mediated proteolysis, Dopaminergic synapse, and Glutamatergic synapse were the most prominent pathways enriched in quantiles with PD miRNA patterns. Messenger RNA (mRNA) transcripts [amyloid precursor protein, APP), α-synuclein (α-syn), Tau, neurofilament, light gene (NF-L), DJ-1/PARK7, Fractalkine and Neurosin] and long non-coding RNAs (RP11-462G22.1 and PCA3) were differentially expressed in CSF exosomes in PD and AD patients. These data demonstrated that CSF exosomal RNA molecules are reliable biomarkers with fair robustness in regard to specificity and sensitivity in differentiating PD from healthy and diseased (AD) controls. PMID:26497684

  1. Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.

    PubMed

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-25

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  2. Chronic Nicotine Exposure Systemically Alters MicroRNA Expression Profiles during Post-embryonic Stages in Caenorhabditis elegans

    PubMed Central

    Taki, Faten A; Pan, Xiaoping; Zhang, Baohong

    2014-01-01

    Tobacco smoking is associated with many diseases. Addiction is of the most notorious tobacco-related syndrome and is majorly attributed to nicotine. In this study, we employed C. elegans as a biological model to systemically investigate the effect of chronic nicotine exposure on microRNA (miRNA) expression profile and their regulated biochemical pathways. Nicotine treatment (20μM and 20mM) was limited to the post-embryonic stage from L1–L4 (~31 hours) period after which worms were collected for genome-wide miRNA profiling. Our results show that nicotine significantly altered the expression patterns of 40 miRNAs. The effect was proportional to the nicotine dose and was expected to have an additive, more robust response. Based on pathway enrichment analyses coupled with nicotine-induced miRNA patterns, we inferred that miRNAs as a system mediates “regulatory hormesis”, manifested in biphasic behavioral and physiological phenotypes. We proposed a model where nicotine addiction is mediated by miRNAs’ regulation of fos-1 and is maintained by epigenetic factors. Thus, our study offers new insights for a better understanding of the sensitivity of early developmental stages to nicotine. PMID:23765240

  3. Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli*

    PubMed Central

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L.; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-01

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  4. Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans.

    PubMed

    Tiwary, B N; Bisen, P S; Sinha, U

    1987-08-01

    We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase. PMID:3312953

  5. Altered Subcellular Localization of the NeuN/Rbfox3 RNA Splicing Factor in HIV-Associated Neurocognitive Disorders (HAND)

    PubMed Central

    Lucas, Calixto-Hope; Calvez, Mathilde; Babu, Roshni; Brown, Amanda

    2013-01-01

    The anti-NeuN antibody has been widely used for over 15 years to unambiguously identify post-mitotic neurons in the central nervous system of a wide variety of vertebrates including mice, rats and humans. In contrast to its widely reported nuclear localization, we found significantly higher NeuN reactivity in the cytoplasm of neurons in brain sections from HIV-infected individuals with cognitive impairment compared to controls. The protein target of anti-NeuN antisera was recently identified as the neuron-specific RNA splicing factor, Rbfox3, but its significance in diseases affecting the brain has not been previously reported. RNA splicing occurs in the nucleus hence, the altered localization of RbFox3 to the cytoplasm may lead to the downregulation of neuronal gene expression. PMID:24215932

  6. EpCAM knockdown alters microRNA expression in retinoblastoma--functional implication of EpCAM regulated miRNA in tumor progression.

    PubMed

    Beta, Madhu; Khetan, Vikas; Chatterjee, Nivedita; Suganeswari, Ganesan; Rishi, Pukhraj; Biswas, Jyotirmay; Krishnakumar, Subramanian

    2014-01-01

    The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes. PMID:25502397

  7. EpCAM Knockdown Alters MicroRNA Expression in Retinoblastoma- Functional Implication of EpCAM Regulated MiRNA in Tumor Progression

    PubMed Central

    Beta, Madhu; Khetan, Vikas; Chatterjee, Nivedita; Suganeswari, Ganesan; Rishi, Pukhraj; Biswas, Jyotirmay; Krishnakumar, Subramanian

    2014-01-01

    The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes. PMID:25502397

  8. Altered microRNA expression profiles in a rat model of spina bifida

    PubMed Central

    Qin, Pan; Li, Lin; Zhang, Da; Liu, Qiu-liang; Chen, Xin-rang; Yang, He-ying; Fan, Ying-zhong; Wang, Jia-xiang

    2016-01-01

    MicroRNAs (miRNAs) are dynamically regulated during neurodevelopment, yet few reports have examined their role in spina bifida. In this study, we used an established fetal rat model of spina bifida induced by intragastrically administering olive oil-containing all-trans retinoic acid to dams on day 10 of pregnancy. Dams that received intragastric administration of all-trans retinoic acid-free olive oil served as controls. The miRNA expression profile in the amniotic fluid of rats at 20 days of pregnancy was analyzed using an miRNA microarray assay. Compared with that in control fetuses, the expression of miRNA-9, miRNA-124a, and miRNA-138 was significantly decreased (> 2-fold), whereas the expression of miRNA-134 was significantly increased (> 4-fold) in the amniotic fluid of rats with fetuses modeling spina bifida. These results were validated using real-time quantitative reverse-transcription polymerase chain reaction. Hierarchical clustering analysis of the microarray data showed that these differentially expressed miRNAs could distinguish fetuses modeling spina bifida from control fetuses. Our bioinformatics analysis suggested that these differentially expressed miRNAs were associated with many cytological pathways, including a nervous system development signaling pathway. These findings indicate that further studies are warranted examining the role of miRNAs through their regulation of a variety of cell functional pathways in the pathogenesis of spina bifida. Such studies may provide novel targets for the early diagnosis and treatment of spina bifida. PMID:27127493

  9. Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

    PubMed Central

    Morrison, J R; Pászty, C; Stevens, M E; Hughes, S D; Forte, T; Scott, J; Rubin, E M

    1996-01-01

    RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692961

  10. Altered microRNA expression profiles in a rat model of spina bifida.

    PubMed

    Qin, Pan; Li, Lin; Zhang, Da; Liu, Qiu-Liang; Chen, Xin-Rang; Yang, He-Ying; Fan, Ying-Zhong; Wang, Jia-Xiang

    2016-03-01

    MicroRNAs (miRNAs) are dynamically regulated during neurodevelopment, yet few reports have examined their role in spina bifida. In this study, we used an established fetal rat model of spina bifida induced by intragastrically administering olive oil-containing all-trans retinoic acid to dams on day 10 of pregnancy. Dams that received intragastric administration of all-trans retinoic acid-free olive oil served as controls. The miRNA expression profile in the amniotic fluid of rats at 20 days of pregnancy was analyzed using an miRNA microarray assay. Compared with that in control fetuses, the expression of miRNA-9, miRNA-124a, and miRNA-138 was significantly decreased (> 2-fold), whereas the expression of miRNA-134 was significantly increased (> 4-fold) in the amniotic fluid of rats with fetuses modeling spina bifida. These results were validated using real-time quantitative reverse-transcription polymerase chain reaction. Hierarchical clustering analysis of the microarray data showed that these differentially expressed miRNAs could distinguish fetuses modeling spina bifida from control fetuses. Our bioinformatics analysis suggested that these differentially expressed miRNAs were associated with many cytological pathways, including a nervous system development signaling pathway. These findings indicate that further studies are warranted examining the role of miRNAs through their regulation of a variety of cell functional pathways in the pathogenesis of spina bifida. Such studies may provide novel targets for the early diagnosis and treatment of spina bifida. PMID:27127493

  11. A potential role for estrogen in cigarette smoke-induced microRNA alterations and lung cancer

    PubMed Central

    Cohen, Amit; Smith, Yoav

    2016-01-01

    Alteration in the expression of microRNAs (miRNAs) is associated with oncogenesis and cancer progression. In this review we aim to suggest that elevated levels of estrogens and their metabolites inside the lungs as a result of cigarette smoke exposure can cause widespread repression of miRNA and contribute to lung tumor development. Anti-estrogenic compounds, such as the components of cruciferous vegetables, can attenuate this effect and potentially reduce the risk of lung cancer (LC) among smokers. PMID:27413713

  12. A potential role for estrogen in cigarette smoke-induced microRNA alterations and lung cancer.

    PubMed

    Cohen, Amit; Burgos-Aceves, Mario Alberto; Smith, Yoav

    2016-06-01

    Alteration in the expression of microRNAs (miRNAs) is associated with oncogenesis and cancer progression. In this review we aim to suggest that elevated levels of estrogens and their metabolites inside the lungs as a result of cigarette smoke exposure can cause widespread repression of miRNA and contribute to lung tumor development. Anti-estrogenic compounds, such as the components of cruciferous vegetables, can attenuate this effect and potentially reduce the risk of lung cancer (LC) among smokers. PMID:27413713

  13. Molecular classification of thyroid lesions by combined testing for miRNA gene expression and somatic gene alterations.

    PubMed

    Wylie, Dennis; Beaudenon-Huibregtse, Sylvie; Haynes, Brian C; Giordano, Thomas J; Labourier, Emmanuel

    2016-04-01

    Multiple molecular markers contribute to the pathogenesis of thyroid cancer and can provide valuable information to improve disease diagnosis and patient management. We performed a comprehensive evaluation of miRNA gene expression in diverse thyroid lesions (n = 534) and developed predictive models for the classification of thyroid nodules, alone or in combination with genotyping. Expression profiling by reverse transcription-quantitative polymerase chain reaction in surgical specimens (n = 257) identified specific miRNAs differentially expressed in 17 histopathological categories. Eight supervised machine learning algorithms were trained to discriminate benign from malignant lesions and evaluated for accuracy and robustness. The selected models showed invariant area under the receiver operating characteristic curve (AUC) in cross-validation (0.89), optimal AUC (0.94) in an independent set of preoperative thyroid nodule aspirates (n = 235), and classified 92% of benign lesions as low risk/negative and 92% of malignant lesions as high risk/positive. Surgical and preoperative specimens were further tested for the presence of 17 validated oncogenic gene alterations in the BRAF, RAS, RET or PAX8 genes. The miRNA-based classifiers complemented and significantly improved the diagnostic performance of the 17-mutation panel (p < 0.001 for McNemar's tests). In a subset of resected tissues (n = 54) and in an independent set of thyroid nodules with indeterminate cytology (n = 42), the optimized ThyraMIR Thyroid miRNA Classifier increased diagnostic sensitivity by 30-39% and correctly classified 100% of benign nodules negative by the 17-mutation panel. In contrast, testing with broad targeted next-generation sequencing panels decreased diagnostic specificity by detecting additional mutations of unknown clinical significance in 19-39% of benign lesions. Our results demonstrate that, independent of mutational status, miRNA expression profiles are strongly

  14. Altered Spinal MicroRNA-146a and the MicroRNA-183 Cluster Contribute to Osteoarthritic Pain in Knee Joints

    PubMed Central

    Li, Xin; Kroin, Jeffrey S; Kc, Ranjan; Gibson, Gary; Chen, Di; Corbett, Grant T; Pahan, Kalipada; Fayyaz, Sana; Kim, Jae-Sung; van Wijnen, Andre J.; Suh, Joon; Kim, Su-Gwan; Im, Hee-Jeong

    2015-01-01

    Objective Examine whether altered expression of microRNAs in central nervous system components is pathologically linked to chronic knee joint pain in osteoarthritis. Methods A surgical animal model for knee joint OA was generated by medial meniscus transection in rats followed by behavioral pain tests. Relationships between pathological changes in knee joint and development of chronic joint pain were examined by histology and imaging analyses. Alterations in microRNAs associated with OA-evoked pain sensation were determined in bilateral lumbar dorsal root ganglia (DRG) and the spinal dorsal horn by microRNA array followed by individual microRNA analyses. Gain- and loss-of-function studies of selected microRNAs (miR-146a and miR-183 cluster) were conducted to identify target pain mediators regulated by these selective microRNAs in glial cells. Results The ipsilateral hind leg displayed significantly increased hyperalgesia after 4 weeks of surgery and sensitivity was sustained for the remainder of the 8 week experimental period (F=341, P<0.001). The development of OA-induced chronic pain was correlated with pathological changes in the knee joints as assessed by histological and imaging analyses. MicroRNA analyses showed that miR-146a and the miR-183 cluster were markedly reduced in the sensory neurons in DRG (L4/L5) and spinal cord from animals experiencing knee joint OA pain. The downregulation of miR-146a and/or the miR-183 cluster in the central compartments (DRG and spinal cord) are closely associated with the upregulation of inflammatory pain mediators. The corroboration between decreases in these signature microRNAs and their specific target pain mediators were further confirmed by gain- and loss-of-function analyses in glia, the major cellular component of the central nervous system (CNS). Conclusion MicroRNA therapy using miR-146a and the miR-183 cluster could be powerful therapeutic intervention for OA in alleviating joint pain and concomitantly regenerating

  15. Altered miRNA processing disrupts brown/white adipocyte determination and associates with lipodystrophy

    PubMed Central

    Mori, Marcelo A.; Thomou, Thomas; Boucher, Jeremie; Lee, Kevin Y.; Lallukka, Susanna; Kim, Jason K.; Torriani, Martin; Yki-Järvinen, Hannele; Grinspoon, Steven K.; Cypess, Aaron M.; Kahn, C. Ronald

    2014-01-01

    miRNAs are important regulators of biological processes in many tissues, including the differentiation and function of brown and white adipocytes. The endoribonuclease dicer is a major component of the miRNA-processing pathway, and in adipose tissue, levels of dicer have been shown to decrease with age, increase with caloric restriction, and influence stress resistance. Here, we demonstrated that mice with a fat-specific KO of dicer develop a form of lipodystrophy that is characterized by loss of intra-abdominal and subcutaneous white fat, severe insulin resistance, and enlargement and “whitening” of interscapular brown fat. Additionally, KO of dicer in cultured brown preadipocytes promoted a white adipocyte–like phenotype and reduced expression of several miRNAs. Brown preadipocyte whitening was partially reversed by expression of miR-365, a miRNA known to promote brown fat differentiation; however, introduction of other miRNAs, including miR-346 and miR-362, also contributed to reversal of the loss of the dicer phenotype. Interestingly, fat samples from patients with HIV-related lipodystrophy exhibited a substantial downregulation of dicer mRNA expression. Together, these findings indicate the importance of miRNA processing in white and brown adipose tissue determination and provide a potential link between this process and HIV-related lipodystrophy. PMID:24983316

  16. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

    PubMed

    Bai, Bing; Hales, Chadwick M; Chen, Ping-Chung; Gozal, Yair; Dammer, Eric B; Fritz, Jason J; Wang, Xusheng; Xia, Qiangwei; Duong, Duc M; Street, Craig; Cantero, Gloria; Cheng, Dongmei; Jones, Drew R; Wu, Zhiping; Li, Yuxin; Diner, Ian; Heilman, Craig J; Rees, Howard D; Wu, Hao; Lin, Li; Szulwach, Keith E; Gearing, Marla; Mufson, Elliott J; Bennett, David A; Montine, Thomas J; Seyfried, Nicholas T; Wingo, Thomas S; Sun, Yi E; Jin, Peng; Hanfelt, John; Willcock, Donna M; Levey, Allan; Lah, James J; Peng, Junmin

    2013-10-01

    Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis. PMID:24023061

  17. PUFA diets alter the microRNA expression profiles in an inflammation rat model

    PubMed Central

    ZHENG, ZHENG; GE, YINLIN; ZHANG, JINYU; XUE, MEILAN; LI, QUAN; LIN, DONGLIANG; MA, WENHUI

    2015-01-01

    Omega-3 and -6 polyunsaturated fatty acids (PUFAs) can directly or indirectly regulate immune homeostasis via inflammatory pathways, and components of these pathways are crucial targets of microRNAs (miRNAs). However, no study has examined the changes in the miRNA transcriptome during PUFA-regulated inflammatory processes. Here, we established PUFA diet-induced autoimmune-prone (AP) and autoimmune-averse (AA) rat models, and studied their physical characteristics and immune status. Additionally, miRNA expression patterns in the rat models were compared using microarray assays and bioinformatic methods. A total of 54 miRNAs were differentially expressed in common between the AP and the AA rats, and the changes in rno-miR-19b-3p, -146b-5p and -183-5p expression were validated using stem-loop reverse transcription-quantitative polymerase chain reaction. To better understand the mechanisms underlying PUFA-regulated miRNA changes during inflammation, computational algorithms and biological databases were used to identify the target genes of the three validated miRNAs. Furthermore, Gene Ontology (GO) term annotation and KEGG pathway analyses of the miRNA targets further allowed to explore the potential implication of the miRNAs in inflammatory pathways. The predicted PUFA-regulated inflammatory pathways included the Toll-like receptor (TLR), T cell receptor (TCR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), mitogen-activated protein kinase (MAPK) and the transforming growth factor-β (TGF-β) pathway. This study is the first report, to the best of our knowledge, on in vivo comparative profiling of miRNA transcriptomes in PUFA diet-induced inflammatory rat models using a microarray approach. The results provide a useful resource for future investigation of the role of PUFA-regulated miRNAs in immune homeostasis. PMID:25672643

  18. Epigallocatechin Gallate-Mediated Alteration of the MicroRNA Expression Profile in 5α-Dihydrotestosterone-Treated Human Dermal Papilla Cells

    PubMed Central

    Shin, Shanghun; Kim, Karam; Lee, Myung Joo; Lee, Jeongju; Choi, Sungjin; Kim, Kyung-Suk; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Youn, Hae Jeong; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan

    2016-01-01

    Background Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. Objective We showed that epigallocatechin gallate (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. Methods We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. Results EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. Conclusion Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs. PMID:27274631

  19. Altered microRNA expression in bovine skeletal muscle with age

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Age dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. Therefore, we performed a comprehensive investigation for miRNAs from 5 differe...

  20. Molecular classification of thyroid lesions by combined testing for miRNA gene expression and somatic gene alterations

    PubMed Central

    Wylie, Dennis; Beaudenon‐Huibregtse, Sylvie; Haynes, Brian C.; Giordano, Thomas J.

    2016-01-01

    Abstract Multiple molecular markers contribute to the pathogenesis of thyroid cancer and can provide valuable information to improve disease diagnosis and patient management. We performed a comprehensive evaluation of miRNA gene expression in diverse thyroid lesions (n = 534) and developed predictive models for the classification of thyroid nodules, alone or in combination with genotyping. Expression profiling by reverse transcription‐quantitative polymerase chain reaction in surgical specimens (n = 257) identified specific miRNAs differentially expressed in 17 histopathological categories. Eight supervised machine learning algorithms were trained to discriminate benign from malignant lesions and evaluated for accuracy and robustness. The selected models showed invariant area under the receiver operating characteristic curve (AUC) in cross‐validation (0.89), optimal AUC (0.94) in an independent set of preoperative thyroid nodule aspirates (n = 235), and classified 92% of benign lesions as low risk/negative and 92% of malignant lesions as high risk/positive. Surgical and preoperative specimens were further tested for the presence of 17 validated oncogenic gene alterations in the BRAF, RAS, RET or PAX8 genes. The miRNA‐based classifiers complemented and significantly improved the diagnostic performance of the 17‐mutation panel (p < 0.001 for McNemar's tests). In a subset of resected tissues (n = 54) and in an independent set of thyroid nodules with indeterminate cytology (n = 42), the optimized ThyraMIR Thyroid miRNA Classifier increased diagnostic sensitivity by 30–39% and correctly classified 100% of benign nodules negative by the 17‐mutation panel. In contrast, testing with broad targeted next‐generation sequencing panels decreased diagnostic specificity by detecting additional mutations of unknown clinical significance in 19–39% of benign lesions. Our results demonstrate that, independent of mutational status, miRNA

  1. LRPPRC mutation suppresses cytochrome oxidase activity by altering mitochondrial RNA transcript stability in a mouse model.

    PubMed

    Xu, Fenghao; Addis, Jane B L; Cameron, Jessie M; Robinson, Brian H

    2012-01-01

    LRPPRC (leucine-rich pentatricopeptide repeat-containing) has been shown to be essential for the maturation of COX (cytochrome c oxidase), possibly by stabilizing RNA transcripts of COXI, COXII and COXIII genes encoded in mtDNA (mitochondrial DNA). We established a mouse 'gene-trap' model using ES cells (embryonic stem cells) in which the C-terminus of LRPPRC has been replaced with a β-geo construct. Mice homozygous for this modification were found to be subject to embryonic lethality, with death before 12.5 dpc (days post-coitum). Biochemical analysis of MEFs (mouse embryonic fibroblasts) isolated from homozygous mutants showed a major decrease in COX activity, with slight reductions in other respiratory chain complexes with mtDNA encoded components. Constructs of LRPPRC containing different numbers of PPRs (pentatricopeptide repeats) were expressed as recombinant proteins and tested for their ability to bind to the COXI mRNA transcript. Full binding required the first 19 PPR motifs. A specific segment of COXI mRNA was identified as the binding target for LRPPRC, encoded by mouse mtDNA nucleotides 5961-6020. These data strongly suggest that LRPPRC is involved in the maturation of COX, and is involved in stabilizing of mitochondrial mRNAs encoding COX transcripts. PMID:21880015

  2. Evolution in vitro of an RNA enzyme with altered metal dependence

    NASA Technical Reports Server (NTRS)

    Lehman, N.; Joyce, G. F.

    1993-01-01

    The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

  3. Altered microRNA expression profile in hepatitis B virus-related hepatocellular carcinoma.

    PubMed

    Park, Keon Uk; Seo, Young-Su; Lee, Yun-Han; Park, Jungwook; Hwang, Ilseon; Kang, Koo Jeong; Nam, Jehyun; Kim, Sang-Woo; Kim, Jin Young

    2015-12-01

    Hepatocellular carcinoma (HCC) is one of the most lethal cancers, accounting for about 600,000 cancer deaths worldwide. Despite aggressive chemotherapy, the 5-year survival rate is less than 30% in the United States. This underscores the need for a better understanding of the molecular and cellular disease features. Many studies have demonstrated that aberrant regulation of microRNA (miRNA) expression plays a critical role in the development of various types of cancers including HCC. Here we analyzed the miRNA expression profile of HCC cases associated with chronic hepatitis B virus infection, one of the major etiologies of HCC. Our study identified 267 miRNAs that were differentially regulated in HCC specimens compared to adjacent normal tissues. We next analyzed putative target genes and the relevant signaling pathways that are regulated by these miRNAs. Our findings support the notion that dysfunction of miRNAs is linked to HCC pathogenesis and may lead to the identification of novel targets for diagnosing and treating HCC. PMID:26190160

  4. Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

    PubMed

    Harding, Louisa B; Schultz, Irvin R; da Silva, Denis A M; Ylitalo, Gina M; Ragsdale, Dave; Harris, Stephanie I; Bailey, Stephanie; Pepich, Barry V; Swanson, Penny

    2016-09-01

    It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to

  5. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease

    PubMed Central

    Folkmann, Andrew W.; Collier, Scott E.; Zhan, Xiaoyan; Aditi; Ohi, Melanie D.; Wente, Susan R.

    2013-01-01

    The conserved multifunctional protein Gle1 regulates gene expression at multiple steps: nuclear messenger (m)RNA export, translation initiation, and translation termination. A GLE1 mutation (FinMajor) is causally linked to human lethal congenital contracture syndrome-1 (LCCS1); however, the resulting perturbations on Gle1 molecular function were unknown. FinMajor results in a Proline-Phenylalanine-Glutamine peptide insertion within the uncharacterized Gle1 coiled-coil domain. Here we find that Gle1 self-associates both in vitro and in living cells via the coiled-coil domain. Electron microscopy reveals high molecular mass Gle1 oligomers form ∼26 nm in diameter disk-shaped particles. With the Gle1-FinMajor protein, these particles are malformed. Moreover, functional assays document a specific requirement for proper Gle1 oligomerization during mRNA export but not for Gle1’s roles in translation. These results identify a novel mechanistic step in Gle1’s mRNA export function at nuclear pore complexes, and directly implicate altered export in LCCS1 disease pathology. PMID:24243016

  6. RNA-binding protein CELF1 promotes tumor growth and alters gene expression in oral squamous cell carcinoma.

    PubMed

    House, Reniqua P; Talwar, Sudha; Hazard, E Starr; Hill, Elizabeth G; Palanisamy, Viswanathan

    2015-12-22

    The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention. PMID:26498364

  7. Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

    PubMed

    Dickens, M; Romero, L M; Cyr, N E; Dunn, I C; Meddle, S L

    2009-10-01

    Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress. PMID:19686439

  8. Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes.

    PubMed

    Mukherjee, A; Koli, S; Reddy, K V R

    2015-09-01

    Mechanistic target of rapamycin (mTOR) is a signal transduction pathway that modulates translation initiation in several animals including mammals. Rapamaycin, an allosteric inhibitor of mTOR pathway, is often used as an immunosuppressive drug following kidney transplantation and causes gonadal dysfunction and defects in spermatogenesis. The molecular mechanism behind rapamycin-mediated testicular dysfunction is not known. We have therefore explored the contribution of rapamycin in mTOR regulation and microRNA (miRNA) expression in mouse spermatocytes, the intermediate stage of spermatogenesis, where meiosis takes place. In the present study, we optimized the isolation of highly pure and viable spermatocytes by flow sorting, treated them with rapamycin, and investigated the expression of mTOR and downstream effector molecules. Western blot and immunocytochemical analysis confirm that rapamycin treatment suppresses mTOR and phopsphorylated P70S6 kinase activities in spermatocytes, but not that of phosphorylated 4E-binding protein 1. Also, rapamycin treatment modulates the expression of several spermatocyte-specific miRNAs. To complement these finding an in vivo study was also performed. In silico prediction of target genes of these miRNAs and their functional pathway analysis revealed that, several of them are involved in crucial biological process, cellular process and catalytic activities. miRNA-transcription factor (TF) network analysis enlisted different TFs propelling the transcription machineries of these miRNAs. In silico prediction followed by quatitative real-time PCR revealed two of these TFs namely, PU.1 and CCCTC binding factor (CTCF) are down and upregulated, respectively, which may be the reason of the altered expression of miRNAs following rapamycin treatment. In conclusion, for the first time, the present study provides insight into how rapamycin regulates mTOR pathway and spermatocyte-specific miRNA expression which in turn, regulate expression of

  9. Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana.

    PubMed

    Jung, Il Lae; Ryu, Moonyoung; Cho, Seok Keun; Shah, Pratik; Lee, Ju Hye; Bae, Hansol; Kim, In Gyu; Yang, Seong Wook

    2015-01-01

    MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism. PMID:25946015

  10. Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana

    PubMed Central

    Cho, Seok Keun; Shah, Pratik; Lee, Ju Hye; Bae, Hansol; Kim, In Gyu; Yang, Seong Wook

    2015-01-01

    MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism. PMID:25946015

  11. Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model

    PubMed Central

    Pfingsten, Jennifer S.; Goodrich, Karen J.; Taabazuing, Cornelius; Ouenzar, Faissal; Chartrand, Pascal; Cech, Thomas R.

    2012-01-01

    SUMMARY In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes its telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt non-homologous end-joining (NHEJ), telomeric gene silencing and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PMID:22365814

  12. Structural model of the p14/SF3b155·branch duplex complex

    PubMed Central

    Schellenberg, Matthew J.; Dul, Erin L.; MacMillan, Andrew M.

    2011-01-01

    Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14•bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein–RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14•duplex interaction must be disrupted before the first step of splicing. PMID:21062891

  13. The first crystal structures of RNA–PNA duplexes and a PNA-PNA duplex containing mismatches—toward anti-sense therapy against TREDs

    PubMed Central

    Kiliszek, Agnieszka; Banaszak, Katarzyna; Dauter, Zbigniew; Rypniewski, Wojciech

    2016-01-01

    PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA–PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA–PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA–RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson–Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA. PMID:26717983

  14. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  15. The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

    SciTech Connect

    Hamrick, Mark W.; Herberg, Samuel; Arounleut, Phonepasong; He, Hong-Zhi; Shiver, Austin; Qi, Rui-Qun; Zhou, Li; Isales, Carlos M.; and others

    2010-09-24

    Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient

  16. Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons

    PubMed Central

    Sakai, Yusuke; Miyauchi, Kenjyo; Kimura, Satoshi; Suzuki, Tsutomu

    2016-01-01

    Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo5U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo5U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo5U and mcmo5U facilitate non-Watson–Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo5U as a major modification in tRNAAla1, tRNASer1, tRNAPro3 and tRNAThr4; by contrast, cmo5U was present primarily in tRNALeu3 and tRNAVal1. In addition, we discovered 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo5Um) as a novel but minor modification in tRNASer1. Terminal methylation frequency of mcmo5U in tRNAPro3 was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo5U to form mcmo5U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo5U contributes to the decoding ability of tRNAAla1. PMID:26681692

  17. Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

    PubMed Central

    Sun, Yan; Borbat, Peter P.; Grigoryants, Vladimir M.; Myers, William K.; Freed, Jack H.; Scholes, Charles P.

    2015-01-01

    Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ∼35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present. PMID:25692594

  18. Epstein-Barr virus growth/latency III program alters cellular microRNA expression

    SciTech Connect

    Cameron, Jennifer E. Fewell, Claire Yin, Qinyan McBride, Jane Wang Xia Lin Zhen

    2008-12-20

    The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

  19. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    PubMed

    Caracciolo, Luca; Barbon, Alessandro; Palumbo, Sara; Mora, Cristina; Toscano, Christopher D; Bosetti, Francesca; Barlati, Sergio

    2011-01-01

    Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  20. Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

    PubMed

    Weissmann, D; van der Laan, S; Underwood, M D; Salvetat, N; Cavarec, L; Vincent, L; Molina, F; Mann, J J; Arango, V; Pujol, J F

    2016-01-01

    Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide. PMID:27576167

  1. The synovial microenvironment of osteoarthritic joints alters RNA-seq expression profiles of human primary articular chondrocytes.

    PubMed

    Lewallen, Eric A; Bonin, Carolina A; Li, Xin; Smith, Jay; Karperien, Marcel; Larson, A Noelle; Lewallen, David G; Cool, Simon M; Westendorf, Jennifer J; Krych, Aaron J; Leontovich, Alexey A; Im, Hee-Jeong; van Wijnen, Andre J

    2016-10-15

    Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain and has limited treatment options. To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed the biological effects of OA-related changes in the synovial microenvironment on chondrocytes embedded within anatomically intact cartilage from joints with different pathological grades by next generation RNA-sequencing (RNA-seq). We determined the transcriptome of primary articular chondrocytes derived from anatomically unaffected knees and ankles, as well as from joints affected by OA. The GALAXY bioinformatics platform was used to facilitate biological interpretations. Comparisons of patient samples by k-means, hierarchical clustering and principal component analyses together reveal that primary chondrocytes exhibit OA grade-related differences in gene expression, including genes involved in cell-adhesion, ECM production and immune response. We conclude that diseased synovial microenvironments in joints with different histopathological OA grades directly alter gene expression in chondrocytes. One ramification of this finding is that anatomically intact cartilage from OA joints is not an ideal source of healthy chondrocytes, nor should these specimens be used to generate a normal baseline for the molecular characterization of diseased joints. PMID:27378743

  2. Deregulated KLF4 Expression in Myeloid Leukemias Alters Cell Proliferation and Differentiation through MicroRNA and Gene Targets

    PubMed Central

    Morris, Valerie A.; Cummings, Carrie L.; Korb, Brendan; Boaglio, Sean

    2015-01-01

    Acute myeloid leukemia (AML) is characterized by increased proliferation and blocked differentiation of hematopoietic progenitors mediated, in part, by altered myeloid transcription factor expression. Decreased Krüppel-like factor 4 (KLF4) expression has been observed in AML, but how decreased KLF4 contributes to AML pathogenesis is largely unknown. We demonstrate decreased KLF4 expression in AML patient samples with various cytogenetic aberrations, confirm that KLF4 overexpression promotes myeloid differentiation and inhibits cell proliferation in AML cell lines, and identify new targets of KLF4. We have demonstrated that microRNA 150 (miR-150) expression is decreased in AML and that reintroducing miR-150 expression induces myeloid differentiation and inhibits proliferation of AML cells. We show that KLF family DNA binding sites are necessary for miR-150 promoter activity and that KLF2 or KLF4 overexpression induces miR-150 expression. miR-150 silencing, alone or in combination with silencing of CDKN1A, a well-described KLF4 target, did not fully reverse KLF4-mediated effects. Gene expression profiling and validation identified putative KLF4-regulated genes, including decreased MYC and downstream MYC-regulated gene expression in KLF4-overexpressing cells. Our findings indicate that decreased KLF4 expression mediates antileukemic effects through regulation of gene and microRNA networks, containing miR-150, CDKN1A, and MYC, and provide mechanistic support for therapeutic strategies increasing KLF4 expression. PMID:26644403

  3. Age-associated miRNA Alterations in Skeletal Muscle from Rhesus Monkeys reversed by caloric restriction

    PubMed Central

    Mercken, Evi M.; Majounie, Elisa; Ding, Jinhui; Guo, Rong; Kim, Jiyoung; Bernier, Michel; Mattison, Julie; Cookson, Mark R.; Gorospe, Myriam; de Cabo, Rafael; Abdelmohsen, Kotb

    2013-01-01

    The levels of microRNAs (miRNAs) are altered under different conditions such as cancer, senescence, and aging. Here, we have identified differentially expressed miRNAs in skeletal muscle from young and old rhesus monkeys using RNA sequencing. In old muscle, several miRNAs were upregulated, including miR-451, miR-144, miR-18a and miR-15a, while a few miRNAs were downregulated, including miR-181a and miR-181b. A number of novel miRNAs were also identified, particularly in old muscle. We also examined the impact of caloric restriction (CR) on miRNA abundance by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis and found that CR rescued the levels of miR-181b and chr1:205580546, and also dampened the age-induced increase in miR-451 and miR-144 levels. Our results reveal that there are changes in expression of known and novel miRNAs with skeletal muscle aging and that CR may reverse some of these changes to a younger phenotype. PMID:24036467

  4. Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions

    PubMed Central

    Hood, Jennifer L.; Morabito, Michael V.; Martinez, Charles R.; Gilbert, James A.; Ferrick, Elizabeth A.; Ayers, Gregory D.; Chappell, James D.; Dermody, Terence S.; Emeson, Ronald B.

    2014-01-01

    Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neuro-tropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo. PMID:24906008

  5. The defective expression of gtpbp3 related to tRNA modification alters the mitochondrial function and development of zebrafish.

    PubMed

    Chen, Danni; Li, Feng; Yang, Qingxian; Tian, Miao; Zhang, Zengming; Zhang, Qinghai; Chen, Ye; Guan, Min-Xin

    2016-08-01

    Human mitochondrial DNA (mtDNA) mutations have been associated with a wide spectrum of clinical abnormalities. However, nuclear modifier gene(s) modulate the phenotypic expression of pathogenic mtDNA mutations. In our previous investigation, we identified the human GTPBP3 related to mitochondrial tRNA modification, acting as a modifier to influence of deafness-associated mtDNA mutation. Mutations in GTPBP3 have been found to be associated with other human diseases. However, the pathophysiology of GTPBP3-associated disorders is still not fully understood. Here, we reported the generation and characterization of Gtpbp3 depletion zebrafish model using antisense morpholinos. Zebrafish gtpbp3 has three isoforms localized at mitochondria. Zebrafish gtpbp3 is expressed at various embryonic stages and in multiple tissues. In particular, the gtpbp3 was expressed more abundantly in adult zebrafish ovary and testis. The expression of zebrafish gtpbp3 can functionally restore the growth defects caused by the mss1/gtpbp3 mutation in yeast. A marked decrease of mitochondrial ATP generation accompanied by increased levels of apoptosis and reactive oxygen species were observed in gtpbp3 knockdown zebrafish embryos. The Gtpbp3 morphants exhibited defective in embryonic development including bleeding, melenin, oedema and curved tails within 5days post fertilization, as compared with uninjected controls. The co-injection of wild type gtpbp3 mRNA partially rescued these defects in Gtpbp3 morphants. These data suggest that zebrafish Gtpbp3 is a structural and functional homolog of human and yeast GTPBP3. The mitochondrial dysfunction caused by defective Gtpbp3 may alter the embryonic development in the zebrafish. In addition, this zebrafish model of mitochondrial disease may provide unique opportunities for studying defective tRNA modification, mitochondrial biogenesis, and pathophysiology of mitochondrial disorders. PMID:27184967

  6. Alterations in MicroRNA Expression Contribute to Fatty Acid–Induced Pancreatic β-Cell Dysfunction

    PubMed Central

    Lovis, Pascal; Roggli, Elodie; Laybutt, D. Ross; Gattesco, Sonia; Yang, Jiang-Yan; Widmann, Christian; Abderrahmani, Amar; Regazzi, Romano

    2008-01-01

    OBJECTIVE—Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic β-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation. RESEARCH DESIGN AND METHODS—We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis. RESULTS—Prolonged exposure of the β-cell line MIN6B1 and pancreatic islets to palmitate causes a time- and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in β-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion. CONCLUSIONS—Our findings suggest that at least part of the detrimental effects of palmitate on β-cells is caused by alterations in the level of specific miRNAs. PMID:18633110

  7. Altered expression of mRNA profiles in blood of early-onset schizophrenia

    PubMed Central

    Xu, Yong; Yao Shugart, Yin; Wang, Guoqiang; Cheng, Zaohuo; Jin, Chunhui; Zhang, Kai; Wang, Jun; Yu, Hao; Yue, Weihua; Zhang, Fuquan; Zhang, Dai

    2016-01-01

    To identify gene expression abnormalities in schizophrenia (SZ), we generated whole-genome gene expression profiles using microarrays on peripheral blood mononuclear cells (PBMCs) from 18 early-onset SZ cases and 12 controls. We detected 84 transcripts differentially expressed by diagnostic status, with 82 genes being upregulated and 2 downregulated. We identified two SZ associated gene coexpression modules (green and red), including 446 genes . The green module is positively correlated with SZ, encompassing predominantly up-regulated genes in SZ; while the red module was negatively correlated with disease status, involving mostly nominally down-regulated genes in SZ. The olfactory transduction pathway was the most enriched pathways for the genes within the two modules. The expression levels of several hub genes, including AKT1, BRCA1, CCDC134, UBD, and ZIC2 were validated using real-time quantitative PCR. Our findings indicate that mRNA coexpression abnormalities may serve as a promising mechanism underlying the development of SZ. PMID:26733343

  8. Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.

    PubMed

    Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming

    2016-04-01

    Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. PMID:26442942

  9. Switch to duplex stainless steels

    SciTech Connect

    Quik, J.M.A.; Geudeke, M.

    1994-11-01

    Duplex stainless steels contain approximately equal proportions of ferrite and austenite. These stainless steels have become an established material of construction in the chemical process industries (CPI). Duplexes offer benefits over austenitic stainless steels and carbon steels because of their higher strength, and good toughness and ductility, in combination with equivalent resistance to general corrosion, as well as better resistance to localized corrosion and stress corrosion cracking. Additionally, duplex materials have thermal-conductivity and thermal-expansion coefficients similar to those of ferritic materials, are tough at low (sub-zero) temperatures, and have a high resistance to erosion and abrasion. In some of the highly corrosive environments encountered in the CPI, the super duplex stainless steels offer cost-effective options not possible with the standard austenitic stainless steels. The initial applications were almost exclusively as heat exchanger tubing in water-cooled service. In recent times, duplex stainless steels have been used in the oil, gas, and chemical industries. Examples include service in sweet and mildly sour corrosive environments, on offshore platforms where weight savings can be realized, and as a replacement for standard austenitic stainless steel in chemical-processing plants.

  10. Altered microRNA expression profiles are involved in resistance to low-dose ionizing radiation in the absence of BMI1 in human dermal fibroblasts.

    PubMed

    Bae, Seunghee; Kim, Karam; Cha, Hwa Jun; Choi, Yeongmin; Shin, Shang Hun; An, In-Sook; Lee, Jae Ho; Song, Jie-Young; Yang, Kwang Hee; Nam, Seon Young; An, Sungkwan

    2014-10-01

    The polycomb group RING finger protein, B-cell‑specific moloney murine leukemia virus integration site 1 (BMI1), has emerged as a key regulator of cell proliferation, cell cycle, cell immortalization, chemoresistance and radioresistance. Although the radioresistant effect of BMI1 has been thoroughly investigated, the effectiveness of this factor on low-dose radiation (LDR) resistance has not been explored. Here, we demonstrate that BMI1 is not critical for altering cell viability or cell growth in response to LDR, but BMI1 changes cellular gene expression profiles in response to LDR. Normal human dermal fibroblasts (NHDFs) stably expressing BMI1 short hairpin RNA (shRNA) did not exhibit changes in cell viability or cell cycle distribution assays following exposure to 0.1 Gy of γ-radiation. However, microRNA (miRNA) microarrays revealed that a lack of BMI1 leads to changes in miRNA expression in response to LDR. Bioinformatics analyses demonstrated that predicted target genes of the altered miRNAs are functionally involved in both negative and positive regulation of cell growth, cell proliferation, cell cycle and apoptosis. Therefore, these results indicate that low radiosensitivity even in the absence of the radioresistant factor BMI1 is related with the altered miRNA expression profiles in NHDF. PMID:25016973

  11. Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells.

    PubMed

    Chen, Yinghua; Mohammed, Arshad; Oubaidin, Maysaa; Evans, Carla A; Zhou, Xiaofeng; Luan, Xianghong; Diekwisch, Thomas G H; Atsawasuwan, Phimon

    2015-07-15

    MicroRNAs (miRs) play an important role in the development and remodeling of tissues through the regulation of large cohorts of extracellular matrix (ECM) genes. The purpose of the present study was to determine the response of miR-29 family expression to loading forces and their effects on ECM gene expression in periodontal ligament cells, the key effector cell population during orthodontic tooth movement. In a comparison between miRs from human periodontal ligament cells (PDLCs) and alveolar bone cells (ABCs) from healthy human subjects, the ABC cohort of miRs was substantially greater than the corresponding PDLC cohort. Cyclic mechanical stretch forces at 12% deformation at 0.1Hz for 24h decreased expression of miR-29 family member miRs about 0.5 fold while 2g/cm(2) compression force for 24h increased miR-29 family member expression in PDLCs 1.8-4 folds. Cyclic stretch up-regulated major ECM genes in PDLCs, such as COL1A1, COL3A1 and COL5A1, while the compression force resulted in a down-regulation of these ECM genes. Direct interactions of miR-29 and Col1a1, Col3a1 and Col5a1 were confirmed using a dual luciferase reporter gene assay. In addition, transient transfection of a miR-29b mimic in mouse PDLCs down-regulated Col1a1, Col3a1 and Col5a1 while the transfection of miR-29b inhibitor up-regulated these genes compared to control transfection indicating that these target ECM genes directly responded to the altered level of miR-29b. These results provided a possible explanation for the effects of the miR-29 family on loaded PDLCS and their roles in extracellular matrix gene expression. PMID:25827718

  12. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

    PubMed Central

    Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

    2015-01-01

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed

  13. Prevention of 5-hydroxytryptamine2C receptor RNA editing and alternate splicing in C57BL/6 mice activates the hypothalamic-pituitary-adrenal axis and alters mood

    PubMed Central

    Bombail, Vincent; Qing, Wei; Chapman, Karen E; Holmes, Megan C

    2014-01-01

    The 5-hydroxytryptamine2C (5-HT)2C receptor is widely implicated in the aetiology of affective and eating disorders as well as regulation of the hypothalamo-pituitary-adrenal axis. Signalling through this receptor is regulated by A-to-I RNA editing, affecting three amino acids in the protein sequence, with unedited transcripts encoding a receptor (INI) that, in vitro, is hyperactive compared with edited isoforms. Targeted alteration (knock-in) of the Htr2c gene to generate ‘INI’ mice with no alternate splicing, solely expressing the full-length unedited isoform, did not produce an overt metabolic phenotype or altered anxiety behaviour, but did display reduced depressive-like and fear-associated behaviours. INI mice exhibited a hyperactive hypothalamo-pituitary-adrenal axis, with increased nadir plasma corticosterone and corticotrophin-releasing hormone expression in the hypothalamus but responded normally to chronic stress and showed normal circadian activity and activity in a novel environment. The circadian patterns of 5-HT2C receptor mRNA and mbii52, a snoRNA known to regulate RNA editing and RNA splicing of 5-HT2C receptor pre-mRNA, were altered in INI mice compared with wild-type control mice. Moreover, levels of 5-HT1A receptor mRNA were increased in the hippocampus of INI mice. These gene expression changes may underpin the neuroendocrine and behavioural changes observed in INI mice. However, the phenotype of INI mice was not consistent with a globally hyperactive INI receptor encoded by the unedited transcript in the absence of alternate splicing. Hence, the in vivo outcome of RNA editing may be neuronal cell type specific. PMID:25257581

  14. Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+†

    PubMed Central

    Zhang, Shuming; Gu, Hong; Chen, Haoyuan; Strong, Emily; Ollie, Edward W.; Kellerman, Daniel; Liang, Danni; Miyagi, Masaru; Anderson, Vernon E.; Piccirilli, Joseph A.; York, Darrin M.; Harris, Michael E.

    2016-01-01

    Solvent D2O and 18O kinetic isotope effects on RNA 2′-O-transphosphorylation catalyzed by Zn2+ demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data. PMID:26859380

  15. Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice

    PubMed Central

    2014-01-01

    Background Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown. Results We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. Conclusions Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response. PMID:24980094

  16. Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis.

    PubMed

    Cantatore, P; Petruzzella, V; Nicoletti, C; Papadia, F; Fracasso, F; Rustin, P; Gadaleta, M N

    1998-08-01

    Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two-fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome. PMID:9720919

  17. Altered miRNA Signature of Developing Germ-cells in Infertile Patients Relates to the Severity of Spermatogenic Failure and Persists in Spermatozoa

    PubMed Central

    Muñoz, Xavier; Mata, Ana; Bassas, Lluís; Larriba, Sara

    2015-01-01

    The aim of this study was to assess the cellular miRNA expression behaviour in testes with spermatogenic failure (SpF). We performed a high-throughput screen of 623 mature miRNAs by a quantitative RT-qPCR-based approach in histologically well-defined testicular samples with spermatogenic disruption at different germ-cell stages, which revealed altered patterns of miRNA expression. We focussed on the differentially expressed miRNAs whose expression correlated with the number of testicular mature germ-cells and described the combined expression values of a panel of three miRNAs (miR-449a, miR-34c-5p and miR-122) as a predictive test for the presence of mature germ-cells in testicular biopsy. Additionally, we determined decreased cellular miRNA content in developing germ-cells of SpF testis; this was more noticeable the earlier the stage of germ-cell differentiation was affected by maturation failure. Furthermore, we showed that the miRNA expression profile in mature sperm from mild SpF patients was widely altered. Our results suggest that the cellular miRNA content of developed germ-cells depends heavily on the efficacy of the spermatogenic process. What is more, spermatozoa that have fulfilled the differentiation process still retain the dysregulated miRNA pattern observed in the developing SpF germ-cells. This altered miRNA molecular signature may have functional implications for the male gamete. PMID:26648257

  18. Cavitation erosion of duplex and super duplex stainless steels

    SciTech Connect

    Kwok, C.T.; Man, H.C.; Cheng, F.T.

    1998-10-05

    Owing to their excellent corrosion resistance, stainless steels are widely used both in the marine, urban water, chemical and food industries. In addition to the corrosive environment, high fluid flow speeds are always encountered for components used in these industries. The cavitation characteristics of S30400 and S31600 austenitic stainless steels and duplex stainless steels were studied in detail by a number of authors. It was generally agreed that S30400 has higher cavitation erosion resistance than that of S31600 due to higher tendency of strain induced martensitic transformation under high impulse of stress. A considerable number of results on stress corrosion cracking characteristics of SDSS and duplex stainless steels have been published but data concerning their cavitation erosion property are extremely rare.

  19. Aggregation of the 35-kDa fragment of TDP-43 causes formation of cytoplasmic inclusions and alteration of RNA processing.

    PubMed

    Che, Mei-Xia; Jiang, Ya-Jun; Xie, Yuan-Yuan; Jiang, Lei-Lei; Hu, Hong-Yu

    2011-07-01

    TAR DNA binding protein of 43 kDa (TDP-43) is a nuclear factor functioning in RNA processing. It is also a major deposited protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (FTLD-U). To understand the mechanism underlying the inclusion body formation and possible functional alteration, we studied some TDP-43 fragments and their effects on RNA processing in cell models. The results show that the 35-kDa fragment of TDP-43 (namely TDP-35, residues 90-414), but not TDP-25A (184-414), is capable of forming cytoplasmic inclusion bodies and altering pre-mRNA splicing. The inclusions formed by TDP-35 can also recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization. The in vitro studies demonstrate that TDP-35, rather than TDP-43 and TDP-25A, is prone to aggregation, and it further serves as a seed to facilitate aggregation of full-length TDP-43. This suggests that fragmentation of TDP-43 leads to cellular redistribution, inclusion body formation, and altered RNA processing, which are implicated in the molecular pathogenesis of ALS and FTLD. PMID:21450909

  20. MicroRNA Expression Is Altered in an Ovalbumin-Induced Asthma Model and Targeting miR-155 with Antagomirs Reveals Cellular Specificity

    PubMed Central

    Plank, Maximilian W.; Maltby, Steven; Tay, Hock L.; Stewart, Jessica; Eyers, Fiona; Hansbro, Philip M.; Foster, Paul S.

    2015-01-01

    MicroRNAs are post-transcriptional regulators of gene expression that are differentially regulated during development and in inflammatory diseases. A role for miRNAs in allergic asthma is emerging and further investigation is required to determine whether they may serve as potential therapeutic targets. We profiled miRNA expression in murine lungs from an ovalbumin-induced allergic airways disease model, and compared expression to animals receiving dexamethasone treatment and non-allergic controls. Our analysis identified 29 miRNAs that were significantly altered during allergic inflammation. Target prediction analysis revealed novel genes with altered expression in allergic airways disease and suggests synergistic miRNA regulation of target mRNAs. To assess the impacts of one induced miRNA on pathology, we targeted miR-155-5p using a specific antagomir. Antagomir administration successfully reduced miR-155-5p expression with high specificity, but failed to alter the disease phenotype. Interestingly, further investigation revealed that antagomir delivery has variable efficacy across different immune cell types, effectively targeting myeloid cell populations, but exhibiting poor uptake in lymphocytes. Our findings demonstrate that antagomir-based targeting of miRNA function in the lung is highly specific, but highlights cell-specificity as a key limitation to be considered for antagomir-based strategies as therapeutics. PMID:26693910

  1. Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

    PubMed

    Ramadoss, Jayanth; Magness, Ronald R

    2012-06-01

    Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional

  2. MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs

    PubMed Central

    2012-01-01

    Background Numerous single nucleotide polymorphisms (SNPs) associated with complex diseases have been identified by genome-wide association studies (GWAS) and expression quantitative trait loci (eQTLs) studies. However, few of these SNPs have explicit biological functions. Recent studies indicated that the SNPs within the 3’UTR regions of susceptibility genes could affect complex traits/diseases by affecting the function of miRNAs. These 3’UTR SNPs are functional candidates and therefore of interest to GWAS and eQTL researchers. Description We developed a publicly available online database, MirSNP (http://cmbi.bjmu.edu.cn/mirsnp), which is a collection of human SNPs in predicted miRNA-mRNA binding sites. We identified 414,510 SNPs that might affect miRNA-mRNA binding. Annotations were added to these SNPs to predict whether a SNP within the target site would decrease/break or enhance/create an miRNA-mRNA binding site. By applying MirSNP database to three brain eQTL data sets, we identified four unreported SNPs (rs3087822, rs13042, rs1058381, and rs1058398), which might affect miRNA binding and thus affect the expression of their host genes in the brain. We also applied the MirSNP database to our GWAS for schizophrenia: seven predicted miRNA-related SNPs (p < 0.0001) were found in the schizophrenia GWAS. Our findings identified the possible functions of these SNP loci, and provide the basis for subsequent functional research. Conclusion MirSNP could identify the putative miRNA-related SNPs from GWAS and eQTLs researches and provide the direction for subsequent functional researches. PMID:23173617

  3. Estrogen-dependent activation of the avian very low density apolipoprotein II and vitellogenin genes. Transient alterations in mRNA polyadenylation and stability early during induction.

    PubMed

    Cochrane, A W; Deeley, R G

    1988-10-01

    Administration of estrogen to egg-laying vertebrates activates unscheduled, hepatic expression of major, egg-yolk protein genes in immature animals and mature males. Two avian yolk protein genes, encoding very low density apolipoprotein II (apoVLDLII) and vitellogenin II, are dormant prior to stimulation with estrogen, but within three days their cognate mRNAs accumulate to become two of the most abundant species in the liver. Accumulation of these mRNAs has been attributed to both induction of transcription and selective, estrogen-dependent mRNA stabilization. We have detected alterations in the size of apoVLDLII mRNA that occur during the first 24 hours that are attributable to a shift in the extent of polyadenylation as steady-state is approached. In vitro transcription assays indicate that primary activation of both genes takes place relatively slowly and that maximal rates of mRNA accumulation occur when the apoVLDLII and vitellogenin II genes are expressed at only 30% and 10% of their fully induced levels, respectively. Transcription data combined with the structural alteration of apoVLDLII mRNA suggest that stability of the two mRNAs may change as steady-state is approached. We have assessed the compatibility of this suggestion with earlier estimates of the kinetics of accumulation of both mRNAs by developing a generally useful algorithm that predicts approach to steady-state kinetics under conditions where both the rate of synthesis and mRNA stability change throughout the accumulation phase of the response. The results predict that the stability of both mRNAs decreases by at least two- to threefold during the approach to steady-state and that, although an additional destabilization of apoVLDLII mRNA may occur following withdrawal of estrogen, the steady-state stability of vitellogenin mRNA is not significantly decreased upon removal of hormone. PMID:3210227

  4. 60 Years of duplex stainless steel applications

    SciTech Connect

    Olsson, J.; Liljas, M.

    1994-12-31

    In this paper the history of wrought duplex stainless steel development and applications is described. Ferritic-austenitic stainless steels were introduced only a few decades after stainless steels were developed. The paper gives details from the first duplex stainless steels in the 1930`s to the super duplex stainless steel development during the 1980`s. During the years much effort has been devoted to production and welding metallurgy as well as corrosion research of the duplex stainless steels. Therefore, duplex stainless steels are to-day established in a wide product range. Numerous important applications are exemplified. In most cases the selection of a duplex steel has been a result of the combination high strength excellent corrosion resistance. In the pulp and paper industry the most interesting use is as vessel material in digesters. For chemical process industry, the duplex steels are currently used in heat exchangers. The largest application of duplex steels exists in the oil and gas/offshore industry. Hundreds of kms of pipelines are installed and are still being installed. An increased use of duplex steels is foreseen in areas where the strength is of prime importance.

  5. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  6. Altered microRNA Expression Profiles and Regulation of INK4A/CDKN2A Tumor Suppressor Genes in Canine Breast Cancer Models.

    PubMed

    Lutful Kabir, Farruk Mohammad; DeInnocentes, Patricia; Bird, Richard Curtis

    2015-12-01

    microRNA (miRNA) expression profiling of cancer versus normal cells may reveal the characteristic regulatory features that can be correlated to altered gene expression in both human and animal models of cancers. In this study, the comprehensive expression profiles of the 277 highly characterized miRNAs from the canine genome were evaluated in spontaneous canine mammary tumor (CMT) models harboring defects in a group of cell cycle regulatory and potent tumor suppressor genes of INK4/CDKN2 family including p16/INK4A, p14ARF, and p15/INK4B. A large number of differentially expressed miRNAs were identified in three CMT cell lines to potentially target oncogenes, tumor suppressor genes and cancer biomarkers. A group of the altered miRNAs were identified by miRNA target prediction tools for regulation of the INK4/CDKN2 family tumor suppressor genes. miRNA-141 was experimentally validated for INK4A 3'-UTR target binding in the CMT cell lines providing an essential mechanism for the post-transcriptional regulation of the INK4A tumor suppressor gene in CMT models. A well-recognized group of miRNAs including miR-21, miR-155, miR-9, miR-34a, miR-143/145, and miR-31 were found to be altered in both CMTs and human breast cancer. These altered miRNAs might serve as potential targets for advancing the development of future therapeutic reagents. These findings further strengthen the validity and use of canine breast cancers as appropriate models for the study of human breast cancers. PMID:26095675

  7. Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway

    PubMed Central

    Sibony, Michal; Abdullah, Majd; Greenfield, Laura; Raju, Deepa; Wu, Ted; Rodrigues, David M.; Galindo-Mata, Esther; Mascarenhas, Heidi; Philpott, Dana J.; Silverberg, Mark S.

    2015-01-01

    Background: Autophagy is implicated in Crohn's disease (CD) pathogenesis. Recent evidence suggests autophagy regulates the microRNA (miRNA)-induced silencing complex (miRISC). Therefore, autophagy may play a novel role in CD by regulating expression of miRISC, thereby altering miRNA silencing. As microbes associated with CD can alter autophagy, we hypothesized that microbial disruption of autophagy affects the critical miRISC component AGO2. Methods: AGO2 expression was assessed in epithelial and immune cells, and intestinal organoids with disrupted autophagy. Microarray technology was used to determine the expression of downstream miRNAs in cells with defective autophagy. Results: Increased AGO2 was detected in autophagy-deficient ATG5−/− and ATG16−/− mouse embryonic fibroblast cells (MEFs) in comparison with wild-type MEFs. Chemical agents and VacA toxin, which disrupt autophagy, increased AGO2 expression in MEFs, epithelial cells lines, and human monocytes, respectively. Increased AGO2 was also detected in ATG7−/− intestinal organoids, in comparison with wild-type organoids. Five miRNAs were differentially expressed in autophagy-deficient MEFs. Pathway enrichment analysis of the differentially expressed miRNAs implicated signaling pathways previously associated with CD. Conclusions: Taken together, our results suggest that autophagy is involved in the regulation of the critical miRISC component AGO2 in epithelial and immune cells and primary intestinal epithelial cells. We propose a mechanism by which autophagy alters miRNA expression, which likely impacts the regulation of CD-associated pathways. Furthermore, as enteric microbial products can manipulate autophagy and AGO2, our findings suggest a novel mechanism by which enteric microbes could influence miRNA to promote disease. PMID:26332312

  8. RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

    PubMed

    Anthony, S J; Darpel, K E; Belaganahalli, M N; Maan, N; Nomikou, K; Sutton, G; Attoui, H; Maan, S; Mertens, P P C

    2011-11-25

    This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication. PMID:21968198

  9. Modulation of Ergot Alkaloids in a Grass-Endophyte Symbiosis by Alteration of mRNA Concentrations of an Ergot Alkaloid Synthesis Gene.

    PubMed

    Mulinti, Prashanthi; Florea, Simona; Schardl, Christopher L; Panaccione, Daniel G

    2016-06-22

    The profile of ergot alkaloids in perennial ryegrass (Lolium perenne) containing the endophytic fungus Epichloë typhina × festucae includes high concentrations of the early pathway metabolites ergotryptamine and chanoclavine-I in addition to the pathway end-product ergovaline. Because these alkaloids differ in activity, we investigated strategies to alter their relative concentrations. An RNAi-based approach reduced the concentration of mRNA from the gene easA, which encodes an enzyme required for a ring closure that separates ergotryptamine and chanoclavine-I from ergovaline. Lower easA mRNA concentrations correlated with lower concentrations of ergovaline and higher concentrations of ergotryptamine and chanoclavine-I. Overexpression of easA led to higher concentrations of ergovaline in leaf blades but not in pseudostems; concentrations of the early pathway metabolites were not altered in overexpression strains. The data indicate that altering the concentration of mRNA from a single gene can change alkaloid flux, but the magnitude of the change was limited and variable. PMID:27248330

  10. Alterations of (/sup 3/H)actinomycin D binding to axotomized dorsal root ganglion cell nuclei: an autoradiographic method to detect changes in chromatin structure and RNA synthesis

    SciTech Connect

    Wells, M.R.

    1984-11-01

    An autoradiographic method was developed to quantify on a comparative basis the binding of (/sup 3/H)actinomycin D (Act D) to the cell nuclei of frozen, unfixed sections of spinal sensory ganglia in rats. After a crush lesion of the sciatic nerve, alterations of (/sup 3/H)Act D binding were found in L5 and L6 dorsal root ganglia which corresponded to changes in RNA synthesis observed in other studies. An increase in Act D binding was seen at 1 to 3 days postoperation, followed by a decrease at 5 to 7 days. By 9 to 11 days a second increase in binding occurred, followed by a decrease at 14 days. Contralateral ganglia exhibited an increase in Act D binding only at 5 days compared with unoperated controls. The timing of the response in axotomized ganglia differed with the distance of the lesion from the cell body. The observed patterns of Act D binding confirm that changes of chromatin structure are closely associated with the alterations of RNA and protein synthesis occurring after axon injury. The method may be useful as an indicator for alterations in RNA synthesis related to changes in chromatin structure in complex tissues.