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https://www.readbyqxmd.com/read/28802260/the-essential-functions-of-krepb4-are-developmentally-distinct-and-required-for-endonuclease-association-with-editosomes
#1
Suzanne M McDermott, Kenneth Stuart
Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei, and several transcripts are differentially edited in bloodstream (BF) and procyclic form (PF) cells correlating with changes in mitochondrial function. Editing is catalyzed by three ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III KREN1, N2, and N3 endonucleases with distinct cleavage specificities. KREPB4 is a common editosome protein that has a degenerate RNase III domain lacking conserved catalytic residues, in addition to zinc-finger and Pumilio/fem-3 mRNA binding factor (PUF) motifs...
August 11, 2017: RNA
https://www.readbyqxmd.com/read/28802259/cis-regulatory-elements-explain-most-of-the-mrna-stability-variation-across-genes-in-yeast
#2
Jun Cheng, Kerstin C Maier, Ziga Avsec, Petra Rus, Julien Gagneur
The stability of mRNA is one of the major determinants of gene expression. Although a wealth of sequence elements regulating mRNA stability has been described, their quantitative contributions to half-life are unknown. Here, we built a quantitative model for Saccharomyces cerevisiae based on functional mRNA sequence features that explains 59% of the half-life variation between genes and predicts half-life at a median relative error of 30%. The model revealed a new destabilizing 3'UTR motif, ATATTC, which we functionally validated...
August 11, 2017: RNA
https://www.readbyqxmd.com/read/28768715/architecture-and-dynamics-of-overlapped-rna-regulatory-networks
#3
Christopher P Lapointe, Melanie A Preston, Daniel Wilinski, Harriet A J Saunders, Zachary T Campbell, Marvin Wickens
A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet, little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities ‒ Puf3p, Puf4p, and Puf5p of S. cerevisiae. Using RNA Tagging, we identified a "super-network" comprised of four sub-networks: Puf3p, Puf4p, and Puf5p sub-networks, and one controlled by both Puf4p and Puf5p...
August 2, 2017: RNA
https://www.readbyqxmd.com/read/28768714/kinetics-of-crpv-and-hcv-ires-mediated-eukaryotic-translation-using-single-molecule-fluorescence-microscopy
#4
Olivier Bugaud, Nathalie Barbier, Hélène Chommy, Nicolas Fiszman, Antoine Le Gall, David Dullin, Matthieu Saguy, Nathalie Westbrook, Karen Perronet, Olivier Namy
Protein synthesis is a complex multi-step process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labelling of the ribosomes are much more complicated. Here we use a non-canonical translation initiation based on internal ribosome entry sites (IRES) and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA...
August 2, 2017: RNA
https://www.readbyqxmd.com/read/28747322/ribonucleoprotein-purification-and-characterization-using-rna-mango
#5
Shyam Ss Panchapakesan, Matthew L Ferguson, Eric J Hayden, Xin Chen, Aaron A Hoskins, Peter J Unrau
The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs...
July 26, 2017: RNA
https://www.readbyqxmd.com/read/28739676/shape-analysis-of-the-htra-rna-thermometer-from-salmonella-enterica
#6
Edric K Choi, Kelsey A Ulanowicz, Yen Anh H Nguyen, Jane K Frandsen, Rachel M Mitton-Fry
RNA thermometers regulate expression of prokaryotic genes involved in heat shock response or in virulence of pathogenic bacteria such as Yersinia, Neisseria, and Salmonella They function through temperature-dependent conformational changes in the mRNAs in which they reside. Most RNA thermometers are found in the 5'-untranslated region (UTR) of the mRNA, where they modulate availability of the ribosome-binding site. We have used SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) assays to systematically characterize the structure and dynamics of the htrA thermometer of Salmonella enterica at single-nucleotide resolution...
July 24, 2017: RNA
https://www.readbyqxmd.com/read/28724535/priming-in-a-permissive-type-i-c-crispr-cas-system-reveals-distinct-dynamics-of-spacer-acquisition-and-loss
#7
Chitong Rao, Denny Chin, Alexander Wilson Ensminger
CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets...
July 19, 2017: RNA
https://www.readbyqxmd.com/read/28724534/m6aviewer-software-for-the-detection-analysis-and-visualization-of-n6-methyl-adenosine-peaks-from-m6a-seq-me-rip-sequencing-data
#8
Agne Antanaviciute, Belinda Baquero-Perez, Christopher M Watson, Sally M Harrison, Carolina Lascelles, Laura Crinnion, Alexander F Markham, David T Bonthron, Adrian Whitehouse, Ian M Carr
Recent methods for transcriptome-wide N6-methyl-adenosine(m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer - a cross-platform application for analysis and visualisation of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches...
July 19, 2017: RNA
https://www.readbyqxmd.com/read/28701522/yeast-non-coding-rna-interaction-network
#9
Simona Panni, Ananth Prakash, Alex Bateman, Sandra Orchard
This article describes the creation of the first expert manually curated non-coding RNA interaction networks for S. cerevisiae. The RNA-RNA and RNA-protein interaction networks have been carefully extracted from the experimental literature and made available through the IntAct database (www.ebi.ac.uk/intact). We provide an initial network analysis and compare their properties to the much larger protein-protein interaction network. We find that the proteins that bind to ncRNAs in the network contain only a small proportion of classical RNA binding domains...
July 12, 2017: RNA
https://www.readbyqxmd.com/read/28701521/translational-repression-of-the-drosophila-nanos-mrna-involves-the-rna-helicase-belle-and-rna-coating-by-me31b-and-trailer-hitch
#10
Michael Götze, Jérémy Dufourt, Christian Ihling, Christiane Rammelt, Stéphanie Pierson, Nagraj Sambrani, Claudia Temme, Andrea Sinz, Martine Simonelig, Elmar Wahle
Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass-spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo...
July 12, 2017: RNA
https://www.readbyqxmd.com/read/28701520/ligand-recognition-and-helical-stacking-formation-are-intimately-linked-in-the-sam-i-riboswitch-regulatory-mechanism
#11
Anne-Marie Dussault, Audrey Dubé, Frédéric Jacques, Jonathan P Grondin, Daniel Lafontaine
Riboswitches are non-coding mRNA elements that control gene expression by altering their structure upon metabolite binding. Although riboswitch crystal structures provide detailed information about RNA-ligand interactions, little knowledge has been gathered to understand how riboswitches modulate gene expression. Here, we study the molecular recognition mechanism of the S-adenosylmethionine SAM-I riboswitch by characterizing the formation of a helical stacking interaction involving the ligand binding process...
July 12, 2017: RNA
https://www.readbyqxmd.com/read/28701519/nineteen-complex-related-factor-prp45-is-required-for-the-early-stages-of-cotranscriptional-spliceosome-assembly
#12
Martina Hálová, Ondřej Gahura, Martin Převorovský, Zdeněk Cit, Marian Novotný, Anna Valentová, Kateřina Abrhámová, František Půta, Petr Folk
Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells...
July 12, 2017: RNA
https://www.readbyqxmd.com/read/28698239/enzymatic-production-of-single-molecule-fish-and-rna-capture-probes
#13
Imre Gaspar, Frank Wippich, Anne Ephrussi
Arrays of singly-labelled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single molecule microscopy analysis and high efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that 1) all steps of the oligonucleotide labelling - including conjugation, enzymatic synthesis and product purification - can be performed in a standard biology laboratory, 2) the process yields >90 %, often >95 % labeled product with minimal carry-over of impurities and 3) the oligonucleotides can be labeled with different dyes or biotin, allowing single molecule FISH or RNA affinity purification, and northern-blot analysis to be performed...
July 11, 2017: RNA
https://www.readbyqxmd.com/read/28694328/the-contribution-of-the-c5-protein-subunit-of-escherichia-coli-ribonuclease-p-to-specificity-for-precursor-trna-is-modulated-by-proximal-5-leader-sequences
#14
Courtney Nicole Niland, David R Anderson, Eckhard Jankowsky, Michael E Harris
Molecular recognition of RNA by RNA processing enzymes and RNA binding proteins often involves the cooperation between multiple subunits. However, the interdependent contributions of RNA and protein subunits to molecular recognition by ribonucleoproteins and protein-protein complexes is relatively unexplored. RNase P is an endonuclease that removes 5' leaders from precursor tRNAs and functions in bacteria as dimer formed by a catalytic RNA subunit (P RNA) and a protein subunit (C5 in E. coli). The P RNA subunit contacts the tRNA body and proximal 5' leader sequences (N(-1) and N(-2)) while C5 binds distal 5' leader sequences (N(-3) to N(-6))...
July 10, 2017: RNA
https://www.readbyqxmd.com/read/28630141/temperature-responsive-mirnas-in-drosophila-orchestrate-adaptation-to-different-ambient-temperatures
#15
Isabel Fast, Charlotte Hewel, Laura Wester, Julia Schumacher, Daniel Gebert, Hans Zischler, Christian Berger, David Rosenkranz
The majority of Drosophila genes are expressed in a temperature-dependent manner, but the way in which small RNAs may contribute to this effect is completely unknown as we currently lack an idea of how small RNA transcriptomes change as a function of temperature. Applying high throughput sequencing techniques complemented by quantitative real-time PCR experiments, we demonstrate that altered ambient temperature induces drastic, but reversible changes in sequence composition and total abundance of both, miRNA- and piRNA populations...
June 19, 2017: RNA
https://www.readbyqxmd.com/read/28630140/three-dimensional-structure-of-the-3-x-tail-of-hepatitis-c-virus-rna-in-monomeric-and-dimeric-states
#16
Angel Cantero-Camacho, Lixin Fan, Yun-Xing Wang, José Gallego
The 3'X domain is a 98-nucleotide region located at the 3' end of hepatitis C virus genomic RNA that plays essential functions in the viral life cycle. It contains an absolutely conserved, 16-base palindromic sequence that promotes viral RNA dimerization, overlapped with a 7-nucleotide tract implicated in a distal contact with a nearby functional sequence. Using small angle X-ray scattering measurements combined with model building guided by NMR spectroscopy, we have studied the stoichiometry, structure and flexibility of domain 3'X and two smaller subdomain sequences as a function of ionic strength, and obtained a three-dimensional view of the full-length domain in its monomeric and dimeric states...
June 19, 2017: RNA
https://www.readbyqxmd.com/read/28625967/rnase-ii-regulates-rnase-ph-and-is-essential-for-cell-survival-during-starvation-and-stationary-phase
#17
Shaheen Sulthana, Ernesto Quesada, Murray P Deutscher
RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary phase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells...
June 16, 2017: RNA
https://www.readbyqxmd.com/read/28611253/n6-methyladenosine-is-required-for-the-hypoxic-stabilization-of-specific-mrnas
#18
Nate J Fry, Brittany A Law, Olga R Ilkayeva, Christopher L Holley, Kyle D Mansfield
Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m6A in the hypoxia response is unknown...
June 13, 2017: RNA
https://www.readbyqxmd.com/read/28606943/role-of-terminator-hairpin-in-biogenesis-of-functional-hfq-binding-srnas
#19
Teppei Morita, Ryo Nishino, Hiroji Aiba
Many Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of 7 or more T residues at the 3' end (Otaka et al. 2011; Ishikawa et al. 2012). Here, we have studied the role of terminator hairpin in biogenesis of sRNAs focusing on SgrS and RyhB in Esherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the polyU tail is shortened...
June 12, 2017: RNA
https://www.readbyqxmd.com/read/28600356/structural-basis-for-ligand-binding-to-the-guanidine-ii-riboswitch
#20
Caroline W Reiss, Scott A Strobel
The guanidine-II riboswitch, also known as mini-ykkC, is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stemloops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cation and functions as a translationally-controlled on-switch. Here we report the structure of a P2 stemloop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1...
June 9, 2017: RNA
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