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https://www.readbyqxmd.com/read/30012570/nano-lc-ms-using-capillary-columns-enables-accurate-quantification-of-modified-ribonucleosides-at-low-femtomol-levels
#1
L Peter Sarin, Sandra D Kienast, Johannes Leufken, Robert L Ross, Agnieszka Dziergowska, Katarzyna Debiec, Elzbieta Sochacka, Patrick A Limbach, Christian Fufezan, Hannes C A Drexler, Sebastian A Leidel
Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes by affecting mRNA translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples...
July 16, 2018: RNA
https://www.readbyqxmd.com/read/30012569/di-tector-defective-interfering-viral-genomes-detector-for-next-generation-sequencing-data
#2
Guillaume Beauclair, Marie Mura, Chantal Combredet, Frederic Tangy, Nolwenn Jouvenet, Anastassia V Komarova
Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbour immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed DI-tector, a user-friendly and freely program that identifies and characterizes cb/sb genomes from Next Generation Sequencing (NGS) data...
July 16, 2018: RNA
https://www.readbyqxmd.com/read/30012568/increasing-the-length-of-poly-pyrimidine-bulges-broadens-rna-conformational-ensembles-with-minimal-impact-on-stacking-energetics
#3
Dawn K Merriman, Jiayi Yuan, Honglue Shi, Ananya Majumdar, Daniel Herschlag, Hashim M Al-Hashimi
Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of non-coding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1, 2, 3, 4 and 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+ (25 mM monovalent salt), helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population < 15%) whereas 1-bulge and 2-bulge motifs adopt predominantly stacked conformations (stacked population > 74%)...
July 16, 2018: RNA
https://www.readbyqxmd.com/read/30006500/adenine-protonation-enables-cyclic-di-gmp-binding-to-cyclic-gamp-sensing-riboswitches
#4
Heiko Keller, A Katharina Weickhmann, Thomas Bock, Jens Wohnert
In certain structural or functional contexts, RNA structures can contain protonated nucleotides. However, a direct role for stably protonated nucleotides in ligand binding and ligand recognition has not yet been demonstrated unambiguously. Previous X-ray structures of c-GAMP binding riboswitch aptamer domains in complex with their near-cognate ligand c-di-GMP suggest that an adenine of the riboswitch either forms two hydrogen bonds to a G nucleotide of the ligand in the unusual enol tautomeric form or that the adenine in its N1 protonated form binds the G nucleotide of the ligand in its canonical keto tautomeric state...
July 13, 2018: RNA
https://www.readbyqxmd.com/read/30006499/snrp-27-the-c-elegans-homolog-of-the-tri-snrnp-27k-protein-has-a-role-in-5-splice-site-positioning-in-the-spliceosome
#5
Alan M Zahler, Lucero E Rogel, Marissa L Glover, Samira Yitiz, J Matthew Ragle, Sol Katzman
The tri-snRNP 27K protein is a component of the human U4/U6-U5 tri-snRNP and contains an N-terminal phosphorylated RS domain. In a forward genetic screen in C. elegans, we previously identified a dominant mutation, M141T, in the highly-conserved C-terminal region of this protein. The mutant allele promotes changes in cryptic 5' splice site choice. To better understand the function of this poorly characterized splicing factor, we performed high-throughput mRNA sequencing analysis on worms containing this dominant mutation...
July 13, 2018: RNA
https://www.readbyqxmd.com/read/30002084/the-importance-of-dna-methylation-of-exons-on-alternative-splicing
#6
Ronna Shayevitch, Dan Askayo, Ifat Keydar, Gil Ast
Alternative splicing contributes to proteome diversity. As splicing occurs co-transcriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of alternative splicing. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To interrogate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA...
July 12, 2018: RNA
https://www.readbyqxmd.com/read/29997263/translation-elongation-and-mrna-stability-are-coupled-through-the-ribosomal-a-site
#7
Gavin Hanson, Najwa Alhusaini, Nathan Morris, Thomas Sweet, Jeff Coller
Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in eukaryotic cells. Previous work by us and others has shown that codon identity exerts a powerful influence on mRNA stability. In Saccharomyces cerevisiae, studies using a handful of reporter mRNAs show that optimal codons increase translation elongation rate, which in turn increase mRNA stability. However, a direct relationship between elongation rate and mRNA stability has not been established across the entire yeast transcriptome...
July 11, 2018: RNA
https://www.readbyqxmd.com/read/29970597/queuosine-modification-protects-cognate-trnas-against-ribonuclease-cleavage
#8
Xiaoyun Wang, Zaneta Matuszek, Yong Huang, Marc Parisien, Qing Dai, Wesley C Clark, Michael H Schwartz, Tao Pan
Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multi-cellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases...
July 3, 2018: RNA
https://www.readbyqxmd.com/read/29970596/the-m6a-reader-protein-ythdc2-interacts-with-the-small-ribosomal-subunit-and-the-5-3-exoribonuclease-xrn1
#9
Jens Kretschmer, Harita Rao, Philipp Hackert, Katherine E Sloan, Claudia Höbartner, Markus T Bohnsack
N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognise and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation or stability of specific mRNAs...
July 3, 2018: RNA
https://www.readbyqxmd.com/read/29959282/an-important-class-of-intron-retention-events-in-human-erythroblasts-is-regulated-by-cryptic-exons-proposed-to-function-as-splicing-decoys
#10
Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G Conboy
During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron...
June 29, 2018: RNA
https://www.readbyqxmd.com/read/29954950/effects-of-flanking-regions-on-hdv-co-transcriptional-folding-kinetics
#11
Yanli Wang, Zhen Wang, Taigang Liu, Sha Gong, Wenbing Zhang
Hepatitis delta virus (HDV) ribozyme performs the self-cleavage activity through folding to a double pseudoknot structure. The folding of functional RNA structures is often coupled with the transcription process. In this work, we developed a new approach for predicting the co-transcriptional folding kinetics of RNA secondary structures with pseudoknots. We theoretically studied the co-transcriptional folding behavior of the 99nt HDV sequence, two upstream flanking sequences and one downstream flanking sequence...
June 28, 2018: RNA
https://www.readbyqxmd.com/read/29954949/the-s-pombe-mitochondrial-transcriptome
#12
Jinjie Shang, Yanmei Yang, Lin Wu, Mengting Zou, Ying Huang
Mitochondrial gene expression is largely controlled through post-transcriptional processes including mitochondrial RNA (mt-RNA) processing, modification, decay and quality control. Defective mitochondrial gene expression results in mitochondrial oxidative phosphorylation (OXPHOS) deficiency and has been implicated in human disease. To fully understand mitochondrial transcription and RNA processing, we performed RNA-seq analyses of mt-RNAs from the fission yeast Schizosaccharomyces pombe. RNA-seq analyses show that the abundance of mt-RNAs varies greatly...
June 28, 2018: RNA
https://www.readbyqxmd.com/read/29950518/identification-and-removal-of-sequencing-artifacts-produced-by-mispriming-during-reverse-transcription-in-multiple-rna-seq-technologies
#13
Haridha Shivram, Vishwanath R Iyer
The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity...
June 27, 2018: RNA
https://www.readbyqxmd.com/read/29941426/biological-classification-with-rna-seq-data-can-alternatively-spliced-transcript-expression-enhance-machine-learning-classifier
#14
Nathan T Johnson, Andi Dhroso, Katelyn J Hughes, Dmitry Korkin
The extent to which the genes are expressed in the cell can be simplistically defined as a function of one or more factors of the environment, lifestyle, and genetics. RNA sequencing (RNA-Seq) is becoming a prevalent approach to quantify gene expression, and is expected to gain better insights to a number of biological and biomedical questions, compared to the DNA microarrays. Most importantly, RNA-Seq allows to quantify expression at the gene and alternative splicing isoform levels. However, leveraging the RNA-Seq data requires development of new data mining and analytics methods...
June 25, 2018: RNA
https://www.readbyqxmd.com/read/29930024/assessing-the-performance-of-mm-pbsa-and-mm-gbsa-methods-8-predicting-binding-free-energies-and-poses-of-protein-rna-complexes
#15
Fu Chen, Huiyong Sun, Junmei Wang, Feng Zhu, Hui Liu, Zhe Wang, Tailong Lei, Youyong Li, Tingjun Hou
Molecular docking provides a computationally efficient way to predict the atomic structural details of protein-RNA interactions (PRI), but accurate prediction of the three-dimensional structures and binding affinities for PRI is still notoriously difficult, partly due to the unreliability of the existing scoring functions for PRI. MM/PBSA and MM/GBSA are more theoretically rigorous than most scoring functions for protein-RNA docking, but their prediction performance for protein-RNA systems remains unclear. Here, we systemically evaluated the capability of MM/PBSA and MM/GBSA to predict the binding affinities and recognize the near-native binding structures for protein-RNA systems with different solvent models and interior dielectric constants (ϵin )...
June 21, 2018: RNA
https://www.readbyqxmd.com/read/29925570/utp14-interaction-with-the-small-subunit-processome
#16
Joshua J Black, Zhaohui Wang, Lisa M Goering, Arlen W Johnson
The SSU Processome (sometimes referred to as 90S) is an early stable intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU Processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU Processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU Processome. Thus, release of U3 is prerequisite for the transition to pre-40S. Our lab proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU Processome protein Utp14...
June 20, 2018: RNA
https://www.readbyqxmd.com/read/29925569/fitness-advantages-conferred-by-the-l20-interacting-rna-cis-regulator-of-ribosomal-protein-synthesis-in-bacillus-subtilis
#17
Arianne M Babina, Darren J Parker, Gene-Wei Li, Michelle M Meyer
In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5'-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in non-coding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined...
June 20, 2018: RNA
https://www.readbyqxmd.com/read/29914874/long-non-coding-rna-repertoire-and-targeting-by-nuclear-exosome-cytoplasmic-exonuclease-and-rnai-in-fission-yeast
#18
Sophie Atkinson, Samuel Marguerat, Danny Bitton, Francois Bachand, Maria Rodriguez-Lopez, Charalampos Rallis, Jean-Francois Lemay, Cristina Cotobal, Michal Malecki, Pawel Smialowski, Juan Mata, Philipp Korber, Jurg Bahler
Long non-coding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive non-coding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyse lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4-times the previously annotated lncRNAs...
June 18, 2018: RNA
https://www.readbyqxmd.com/read/29903832/influenza-a-virus-derived-sirnas-increase-in-the-absence-of-ns1-yet-fail-to-inhibit-virus-replication
#19
Kevin Tsai, David G Courtney, Edward M Kennedy, Bryan R Cullen
While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaute 2, fails to enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells...
June 14, 2018: RNA
https://www.readbyqxmd.com/read/29895677/microprocessor-dependent-processing-of-splice-site-overlapping-microrna-exons-does-not-result-in-changes-in-alternative-splicing
#20
Giulia Pianigiani, Danilo Licastro, Paola Fortugno, Daniele Castiglia, Ivana Petrovic, Franco Pagani
MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These Splice site Overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in non-coding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons...
June 12, 2018: RNA
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