Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels.
Abstract Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample i...
Source: RNA - May 21, 2020 Category: Genetics & Stem Cells Authors: Zhang W, Xu R, Matuszek Ż, Cai Z, Pan T Tags: RNA Source Type: research

Revisiting the window of opportunity for co-transcriptional splicing in budding yeast.
We reported previously that, in budding yeast, transcription rate affects both the efficiency and fidelity of pre-mRNA splicing, especially of ribosomal protein transcripts. Here, we report that the majority of ribosomal protein transcripts with non-consensus 5' splice sites are spliced less efficiently when transcription is faster, and more efficiently with slower transcription. These results support the "window of opportunity" model, and we suggest a possible mechanism to explain these findings. PMID: 32439718 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - May 21, 2020 Category: Genetics & Stem Cells Authors: Aslanzadeh V, Beggs JD Tags: RNA Source Type: research

Pseudouridine-mediated stop codon read-through in S. cerevisiae is sequence context-independent.
Abstract We have previously shown that when the uridine of a stop codon (UAA, UAG or UGA) is pseudouridylated, the ribosome reads through the modified stop codon. However, it is not clear as to whether or not the pseudouridine (Ψ)-mediated read-through is dependent on the sequence context of mRNA. Here, we use several different approaches and the yeast system to address this question. We show that when a stop codon (pre-mature termination codon, PTC) is introduced into the coding region of a reporter mRNA at several different positions (with different sequence contexts) and pseudouridylated, we detect similar ...
Source: RNA - May 20, 2020 Category: Genetics & Stem Cells Authors: Adachi H, Yu YT Tags: RNA Source Type: research

Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA.
Abstract The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairp...
Source: RNA - May 19, 2020 Category: Genetics & Stem Cells Authors: Brillet K, Martinez-Zapien D, Bec G, Ennifar E, Dock-Bregeon AC, Lebars I Tags: RNA Source Type: research

Adaptor protein RapZ activates endoribonuclease RNase E by protein-protein interaction to cleave a small regulatory RNA.
In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form. PMID: 32424019 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - May 18, 2020 Category: Genetics & Stem Cells Authors: Durica-Mitic S, Göpel Y, Amman F, Görke B Tags: RNA Source Type: research

Parallel poly(A) homo- and hetero-duplex formation detection with an adapted DNA nanoswitch technique.
Abstract Polyriboadenylic (poly(rA)) strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic conditions are held together by A+-A+ base-pairing also involving base interactions with the phosphate backbone. Traditional UV-melting studies of parallel poly(A) duplexes have typically examined homo-duplex formation of a single nucleic acid species in solution. We have adapted a technique utilizing a DNA nanoswitch that detects interaction of two different strands either with similar or differing lengths or modifications. Our method detected ...
Source: RNA - May 15, 2020 Category: Genetics & Stem Cells Authors: Pickard MAG, Brylow KB, Cisco LA, Anecelle MR, Pershun ML, Chandrasekaran AR, Halvorsen K, Gleghorn ML Tags: RNA Source Type: research

Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination.
Abstract DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nts upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the lev...
Source: RNA - May 15, 2020 Category: Genetics & Stem Cells Authors: Ojha S, Jain C Tags: RNA Source Type: research

Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation.
In this study, we found that c-Jun N-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2-PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution...
Source: RNA - May 13, 2020 Category: Genetics & Stem Cells Authors: Chen CA, Strouz K, Huang KL, Shyu AB Tags: RNA Source Type: research

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look.
Abstract As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of th...
Source: RNA - May 12, 2020 Category: Genetics & Stem Cells Authors: Rangan R, Zheludev IN, Das R Tags: RNA Source Type: research

Repeat RNAs associate with replication forks and post-replicative DNA.
Abstract Non-coding RNA has a proven ability to direct and regulate chromatin modifications by acting as scaffolds between DNA and histone-modifying complexes. However, it is unknown if ncRNA plays any role in DNA replication and epigenome maintenance, including histone eviction and re-instalment of histone-modifications after genome duplication. Isolation of nascent chromatin has identified a large number of RNA-binding proteins in addition to unknown components of the replication and epigenetic maintenance machinery. Here, we isolated and characterized long and short RNAs associated with nascent chromatin at act...
Source: RNA - May 11, 2020 Category: Genetics & Stem Cells Authors: Gylling HM, Gonzalez-Aguilera C, Smith MA, Kaczorowski DC, Groth A, Lund AH Tags: RNA Source Type: research

tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA.
In conclusion, we uncover the mechanistic basis for the preferred modification order in the E. coli tRNA elbow region. PMID: 32385137 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - May 8, 2020 Category: Genetics & Stem Cells Authors: Schultz SK, Kothe U Tags: RNA Source Type: research

Specificity in the Biosynthesis of the Universal tRNA Nucleoside N6-Threonylcarbamoyl Adenosine (t6A) - TsaD is the Gatekeeper.
Abstract N6-threonylcarbamoyl adenosine (t6A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t6A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO2, and TsaD catalyzing the transfer of t...
Source: RNA - May 8, 2020 Category: Genetics & Stem Cells Authors: Swinehart WE, Deutsch CW, Sarachan KL, Luthra A, Bacusmo JM, de Cr Eacutecy-Lagard VE, Swairjo MA, Agris PF, Iwata-Reuyl D Tags: RNA Source Type: research

Nop9 recognizes structured and single-stranded RNA elements of pre-ribosomal RNA.
Abstract Nop9 is an essential factor in the processing of pre-ribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its...
Source: RNA - May 5, 2020 Category: Genetics & Stem Cells Authors: Zhang J, Teramoto T, Qiu C, Wine RN, Gonzalez LE, Baserga SJ, Hall TM Tags: RNA Source Type: research

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.
L, Szachniuk M, Tian S, Wang J, Wang J, Watkins AM, Wiedemann J, Xiao Y, Xu X, Yesselman JD, Zhang D, Zhang Y, Zhang Z, Zhao C, Zhao P, Zhou Y, Zok T, Zyla A, Ren A, Batey RT, Golden BL, Huang L, Lilley DM, Liu Y, Patel DJ, Westhof E Abstract RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of six RNA sequences: four structures of nucleolytic ribozymes and two of riboswitches. Systema...
Source: RNA - May 5, 2020 Category: Genetics & Stem Cells Authors: Miao Z, Adamiak RW, Antczak M, Boniecki MJ, Bujnicki JM, Chen SJ, Cheng CY, Cheng Y, Chou FC, Das R, Dokholyan NV, Ding F, Geniesse C, Jiang Y, Joshi A, Krokhotin A, Magnus M, Mailhot O, Major F, Mann TH, Piatkowski P, Pluta R, Popenda M, Sarzynska J, Sun Tags: RNA Source Type: research

Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection.
Abstract The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be m...
Source: RNA - May 1, 2020 Category: Genetics & Stem Cells Authors: Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R Tags: RNA Source Type: research

A robust and versatile method for production and purification of large-scale RNA samples for structural biology.
Abstract Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs, of unknown function and structure, is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity towards ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro trans...
Source: RNA - April 30, 2020 Category: Genetics & Stem Cells Authors: Karlsson H, Baronti L, Petzold K Tags: RNA Source Type: research

5-Methylcytosine Modification of an Epstein-Barr Virus Noncoding RNA Decreases its Stability.
Abstract Many cellular noncoding RNAs contain chemically modified nucleotides that are essential for their function. The Epstein-Barr virus expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2. To examine whether these viral RNAs contain modified nucleotides, we purified native EBERs from EBV-infected cells and performed mass spectrometry analysis. While EBER2 contains no modified nucleotides at stoichiometric amounts, EBER1 was found to carry 5-methylcytosine (m5C) modification. Bisulfite sequencing indicated that a single cytosine of EBER1 is methylated in ~95% of molecules, an...
Source: RNA - April 30, 2020 Category: Genetics & Stem Cells Authors: Henry BA, Kanarek JP, Kotter A, Helm M, Lee N Tags: RNA Source Type: research

A bacterial riboswitch class senses xanthine and uric acid to regulate genes associated with purine oxidation.
Abstract Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the 'NMT1 motif', which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes,...
Source: RNA - April 28, 2020 Category: Genetics & Stem Cells Authors: Yu D, Breaker RR Tags: RNA Source Type: research

Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.
We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with sub-nanogram amounts of input cDNA. Its efficiency, high sensitivity, ease of implementation, and low cost (~$5 per sample) re...
Source: RNA - April 28, 2020 Category: Genetics & Stem Cells Authors: Prezza G, Heckel T, Dietrich S, Homberger C, Westermann AJ, Vogel J Tags: RNA Source Type: research

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts.
Abstract The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' s...
Source: RNA - April 20, 2020 Category: Genetics & Stem Cells Authors: Parra MK, Zhang W, Vu J, DeWitt MA, Conboy JG Tags: RNA Source Type: research

Aminoacyl-tRNA Synthetases.
Abstract The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that play a critical role in protein synthesis, pairing tRNAs with their cognate amino acids according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of non-cognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress condition...
Source: RNA - April 17, 2020 Category: Genetics & Stem Cells Authors: Rubio Gomez MA, Ibba M Tags: RNA Source Type: research

Phased nucleotide inserts for sequencing low-diversity RNA samples from in vitro selection experiments.
Abstract In vitro selection combined with high-throughput sequencing is a powerful experimental approach with broad application in the engineering and characterization of RNA molecules. The diverse pools of starting sequences used for selection are often flanked by fixed sequences used as primer binding sites. The low nucleotide diversity of these sequences causes reduced output and poor read quality on Illumina platforms due to complications with fluorescence imaging algorithms. A common method to alleviate this problem is the addition of fragmented bacteriophage PhiX genome. The increased diversity allows for se...
Source: RNA - April 16, 2020 Category: Genetics & Stem Cells Authors: Bendixsen DP, Roberts J, Townshend B, Hayden EJ Tags: RNA Source Type: research

Metabolic labelling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels.
Abstract Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are th...
Source: RNA - April 15, 2020 Category: Genetics & Stem Cells Authors: Tiana M, Acosta-Iborra B, Hernandez-Sierra R, Galiana-Ribote C, Fernandez-Moreno MA, Jimenez B, Del Peso L Tags: RNA Source Type: research

Structure and ligand binding of the ADP-binding domain of the NAD+ riboswitch.
Abstract The nadA motif is the first known NAD+-dependent riboswitch, comprising two similar tandem bulged stem-loop structures. We have determined the structure of the 5' domain 1 of the riboswitch. It has three coaxial helical segments, separated by an ACANCCCC bulge and by an internal loop, with a tertiary contact between them that includes two C:G base pairs. We have determined the structure with a number of ligands related to NADH, but in each case only the ADP moiety is observed. The adenosine adopts an anti conformation, and forms multiple hydrogen bonds across the width of the sugar edge of the penultimate...
Source: RNA - April 15, 2020 Category: Genetics & Stem Cells Authors: Huang L, Wang J, Lilley D Tags: RNA Source Type: research

Requirement for cleavage factor IIm in the control of alternative polyadenylation in breast cancer cells.
Abstract Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11, the latter protein binds to RNA and the RNA polymerase II carboxy terminal domain. Here, we used siRNA mediated knock-down and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology ter...
Source: RNA - April 15, 2020 Category: Genetics & Stem Cells Authors: Turner RE, Henneken LM, Liem-Weits M, Harrison PF, Swaminathan A, Vary R, Nikolic I, Simpson KJ, Powell DR, Beilharz T, Dichtl B Tags: RNA Source Type: research

An ancient evolutionary connection between Ribonuclease A and EndoU families.
Abstract The ribonuclease A family of proteins is well studied from the biochemical and biophysical points of view, but its evolutionary origins are obscure, as no sequences homologous to this family have been reported outside of vertebrates. Recently, the spatial structure of the ribonuclease domain from a bacterial polymorphic toxin was shown to be closely similar to the structure of vertebrate ribonuclease A. The absence of sequence similarity between the two structures prompted a speculation of convergent evolution of bacterial and vertebrate ribonuclease A-like enzymes. We show that bacterial and homologous a...
Source: RNA - April 13, 2020 Category: Genetics & Stem Cells Authors: Mushegian A, Sorokina I, Eroshkin A, Dlakić M Tags: RNA Source Type: research

Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols.
Abstract In recent years RNA-sequencing (RNA-seq) has emerged as a powerful technology for transcriptome profiling. For a given gene, the number of mapped reads is not only dependent on its expression level and gene length, but also the sequencing depth. To normalize these dependencies, RPKM (Reads Per Kilobase of transcript per Million reads mapped) and TPM (Transcripts Per Million) were used to measure gene or transcript expression levels. A common misconception is that RPKM and TPM values are already normalized, and thus should be comparable across samples or RNA-seq projects. However, RPKM and TPM represent th...
Source: RNA - April 13, 2020 Category: Genetics & Stem Cells Authors: Zhao S, Ye Z, Stanton R Tags: RNA Source Type: research

Evaluating DCA-based method performances for RNA contact prediction by a well-curated dataset.
Abstract RNA molecules play many pivotal roles in a cell that are still not fully understood. Any detailed understanding of RNA function requires knowledge of its three-dimensional structure, yet experimental RNA structure resolution remains demanding. Recent advances in sequencing provide unprecedented amounts of sequence data that can be statistically analysed by methods such as Direct Coupling Analysis (DCA) to determine spatial proximity or contacts of specific nucleic acid pairs, which improve the quality of structure prediction. To quantify this structure prediction improvement, we here present a well curate...
Source: RNA - April 10, 2020 Category: Genetics & Stem Cells Authors: Pucci F, Zerihun MB, Peter EK, Schug A Tags: RNA Source Type: research

Sulfonated inhibitors of the RNA editing ligases validate the essential role of the MRP1/2 proteins in kinetoplastid RNA editing.
In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the ...
Source: RNA - April 10, 2020 Category: Genetics & Stem Cells Authors: Mehta V, Moshiri H, Srikanth A, Kala S, Lukes J, Salavati R Tags: RNA Source Type: research

Splicing conservation signals in plant long non-coding RNAs.
Abstract Long non-coding RNAs (lncRNAs) have recently emerged as prominent regulators of gene expression in eukaryotes. LncRNAs often drive the modification and maintenance of gene activation or gene silencing states via chromatin conformation rearrangements. In plants, lncRNAs have been shown to participate in gene regulation, and are essential to processes such as vernalization and photomorphogenesis. Despite their prominent functions only over a dozen lncRNAs have been experimentally and functionally characterized. Similar to its animal counterparts, the rates of sequence divergence are much higher in plant lnc...
Source: RNA - April 2, 2020 Category: Genetics & Stem Cells Authors: Corona-Gomez JA, Garcia-Lopez IJ, Stadler PF, Fernandez-Valverde SL Tags: RNA Source Type: research

A rapid and simple quantitative method for specific Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR): Application to the detection of viral subgenomic RNAs.
Abstract RNAs that are 5'-truncated versions of a longer RNA, but share the same 3' terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to - and amplify - the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost and specific detection of these truncated RNAs, we report Detection of S...
Source: RNA - April 1, 2020 Category: Genetics & Stem Cells Authors: Kanodia P, Prasanth KR, Roa-Linares VC, Bradrick SS, Garcia-Blanco MA, Miller WA Tags: RNA Source Type: research

Corrigendum: Strategies for genetic inactivation of long noncoding RNAs in zebrafish.
PMID: 32179707 [PubMed - in process] (Source: RNA)
Source: RNA - March 19, 2020 Category: Genetics & Stem Cells Authors: Lavalou P, Eckert H, Damy L, Constanty F, Majello S, Bitetti A, Graindorge A, Shkumatava A Tags: RNA Source Type: research

YTHDF2 destabilizes m6A-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells.
Abstract N6-methyladenosine (m6A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m6A-specific RNA binding proteins. Despite accumulating evidence that m6A plays an important role in modulating pluripotency, the influence of m6A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m6A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m6A-modified transcripts associated with neural development...
Source: RNA - March 13, 2020 Category: Genetics & Stem Cells Authors: Heck AM, Russo J, Wilusz J, Osborne Nishimura E, Wilusz CJ Tags: RNA Source Type: research

Former Orphan Riboswitches Reveal Unexplored Areas of Bacterial Metabolism, Signaling and Gene Control Processes.
Abstract Comparative sequence analyses have been used to discover numerous classes of structured noncoding RNAs, some of which are riboswitches that specifically recognize small-molecule or elemental ion ligands and influence expression of adjacent downstream genes. Determining the correct identity of the ligand for a riboswitch candidate typically is aided by an understanding of the genes under its regulatory control. Riboswitches whose ligands were straightforward to identify have largely been associated with well-characterized metabolic pathways, such as coenzyme or amino acid biosynthesis. Riboswitch candidate...
Source: RNA - March 12, 2020 Category: Genetics & Stem Cells Authors: Sherlock M, Breaker RR Tags: RNA Source Type: research

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.
Abstract Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that n...
Source: RNA - March 6, 2020 Category: Genetics & Stem Cells Authors: Pichkur EB, Paleskava A, Tereshchenkov AG, Kasatsky P, Komarova E, Shiriaev D, Bogdanov A, Dontsova OA, Osterman I, Sergiev PV, Polikanov YS, Myasnikov AG, Konevega AL Tags: RNA Source Type: research

5' tRNA halves are highly expressed in the primate hippocampus and might sequence-specifically regulate gene expression.
Abstract Fragments of mature tRNAs have long been considered as mere degradation products without physiological function. However, recent reports show that tRNA-derived small RNAs (tsRNAs) play prominent roles in diverse cellular processes across a wide spectrum of species. Contrasting the situation in other small RNA pathways the mechanisms behind these effects appear more diverse, more complex and are generally less well understood. In addition, surprisingly little is known about the expression profiles of tsRNAs across different tissues and species. Here, we provide an initial overview of tsRNA expression in di...
Source: RNA - March 6, 2020 Category: Genetics & Stem Cells Authors: Jehn J, Treml J, Wulsch S, Ottum B, Erb V, Hewel C, Kooijmans RN, Wester L, Fast I, Rosenkranz D Tags: RNA Source Type: research

Reciprocal monoallelic expression of ASAR lncRNA genes controls replication timing of human chromosome 6.
In this report, we describe a second lncRNA gene (ASAR6-141) on human chromosome 6 that when disrupted results in delayed replication timing in cis. ASAR6-141 is subject to random monoallelic expression and asynchronous replication, and is expressed from the opposite chromosome 6 homolog as ASAR6. ASAR6-141 RNA, like ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the chromosome territory where it is transcribed. Three classes of cis-acting elements control proper chromosome function in mammals: origins of replication, centromeres; and telomeres, which are responsible for replication, segregat...
Source: RNA - March 6, 2020 Category: Genetics & Stem Cells Authors: Heskett M, Smith LG, Spellman P, Thayer M Tags: RNA Source Type: research

Muscleblind-like 2 controls the hypoxia response of cancer cells.
Abstract Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 in...
Source: RNA - March 3, 2020 Category: Genetics & Stem Cells Authors: Fischer S, Di Liddo A, Taylor K, Gerhardus JS, Sobczak K, Zarnack K, Weigand JE Tags: RNA Source Type: research

Transitions Between the Steps of Forward and Reverse Splicing of Group IIC introns.
Abstract Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependen...
Source: RNA - March 3, 2020 Category: Genetics & Stem Cells Authors: Smathers CM, Robart AR Tags: RNA Source Type: research

Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone.
Abstract The functions of eukaryotic mRNAs are characterized by intramolecular interactions between their 5' and 3' ends. Here, we have addressed the question whether such 5'-3' interactions are established by diffusion-controlled encounter of the ends 'through solution' or by some type of scanning along the RNA backbone. For this purpose, we used an in vitro translation system derived from Drosophila embryo extract that displays two types of 5'-3' interactions: cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug Recognition Elements (SREs) in the 3' UTR. Chimeric RNAs wer...
Source: RNA - February 28, 2020 Category: Genetics & Stem Cells Authors: Kluge F, Goetze M, Wahle E Tags: RNA Source Type: research

PIWI-piRNA pathway-mediated transposable element repression in Hydra somatic stem cells.
Abstract Transposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway we isolated piRNAs from Hydra that...
Source: RNA - February 19, 2020 Category: Genetics & Stem Cells Authors: Teefy BB, Siebert S, Cazet JF, Lin H, Juliano CE Tags: RNA Source Type: research

Axon micro-dissection and transcriptome profiling reveals the in vivo RNA content of fully differentiated myelinated motor axons.
Abstract Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral ...
Source: RNA - February 12, 2020 Category: Genetics & Stem Cells Authors: Farias J, Holt CE, Sotelo JR, Sotelo-Silveira JR Tags: RNA Source Type: research

A Comparative Study of Small Molecules Targeting eIF4A.
Abstract The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity - generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anti-cancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F and sever...
Source: RNA - February 3, 2020 Category: Genetics & Stem Cells Authors: Naineni SK, Itoua Maiga R, Cencic R, Putnam AA, Amador LA, Rodriguez AD, Jankowsky E, Pelletier J Tags: RNA Source Type: research

Kiyoshi Nagai (1949-2019).
n R PMID: 31949100 [PubMed - in process] (Source: RNA)
Source: RNA - January 19, 2020 Category: Genetics & Stem Cells Authors: Lührmann R Tags: RNA Source Type: research

A comparative analysis of ADAR mutant mice reveals site-specific regulation of RNA editing.
Abstract Adenosine-to-inosine RNA editing is an essential posttranscriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs, editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, no comprehensive and quantitative studies have been undertaken to determine how specific ADARs contribute to conserved sites in vivo Here, we amplified each RNA region with editing site(s) separately and combined these for deep sequencing. Th...
Source: RNA - January 15, 2020 Category: Genetics & Stem Cells Authors: Costa Cruz PH, Kato Y, Nakahama T, Shibuya T, Kawahara Y Tags: RNA Source Type: research

Tissue-specific changes in the RNA structurome mediate salinity response in Arabidopsis.
Abstract Little is known concerning the effects of abiotic factors on in vivo RNA structures. We applied Structure-seq to assess the in vivo mRNA structuromes of Arabidopsis thaliana under salinity stress, which negatively impacts agriculture. Structure-seq utilizes dimethyl sulfate reactivity to identify As and Cs that lack base pairing or protection. Salt stress refolded transcripts differentially in root vs. shoot, evincing tissue-specificity of the structurome. Both tissues exhibited an inverse correlation between salt stress-induced changes in transcript reactivity and changes in abundance, with stress-relate...
Source: RNA - January 14, 2020 Category: Genetics & Stem Cells Authors: Tack DC, Su Z, Yu Y, Bevilacqua PC, Assmann SM Tags: RNA Source Type: research

Substrate analogs that trap the 2'-phospho-ADP-ribosylated RNA intermediate of the Tpt1 (tRNA 2'-phosphotransferase) reaction pathway.
We report that replacing the NMN ribose of NAD+ with 2'-fluoroarabinose (thereby eliminating the ribose O2'' nucleophile) results in durable trapping of RNA-2'-phospho-(ADP-fluoroarabinose) as a "dead-end" product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2''F-NAD+ as a substrate. PMID: 31932322 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - January 13, 2020 Category: Genetics & Stem Cells Authors: Dantuluri S, Abdullahu L, Munir A, Katolik A, Damha MJ, Shuman S Tags: RNA Source Type: research

Hexamer phasing governs transcription initiation in the 3'-leader of Ebola virus.
Abstract The genomic, bipartite replication promoter of Ebola virus (EBOV) consists of elements 1 (PE1) and 2 (PE2). PE1 (55 nt at the 3'-terminus) is separated from PE2 (harboring eight 3'-UN5 hexamers) by the transcription start sequence (TSS) of the first nucleoprotein (NP) gene plus a spacer sequence. Insertions or deletions in the spacer were reported to support genome replication if comprising 6 or 12, but not 1/2/3/5/9 nucleotides. This gave rise to formulation of the "rule of 6" for the EBOV replication promoter. Here, we studied the impact of such hexamer phasing on viral transcription using a s...
Source: RNA - January 10, 2020 Category: Genetics & Stem Cells Authors: Bach S, Biedenkopf N, Gruenweller A, Becker S, Hartmann RK Tags: RNA Source Type: research

The presence of the ACA box in the archaeal H/ACA guide RNA allows atypical pseudouridylation.
This study will help elucidate the conditions for production of non-constitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing. PMID: 31919243 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - January 9, 2020 Category: Genetics & Stem Cells Authors: Majumder M, Mukhopadhyay S, Kharel P, Gupta R Tags: RNA Source Type: research

eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide.
Abstract The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It w...
Source: RNA - January 8, 2020 Category: Genetics & Stem Cells Authors: Zhou F, Zhang H, Kulkarni S, Lorsch JR, Hinnebusch AG Tags: RNA Source Type: research