The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).

• MeSH
• Comovirus genetika   MeSH
• DNA řízené RNA-polymerasy genetika   MeSH
• fylogeneze MeSH
• konzervovaná sekvence MeSH
• léčivé rostliny MeSH
• molekulární sekvence - údaje MeSH
• Raphanus genetika   virologie   MeSH
• RNA virová genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza RNA MeSH
• sekvenční homologie aminokyselin MeSH
• viry mozaiky genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• RNA virová MeSH

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

• Klíčová slova
• NudC, RNA capping, RNA polymerase, RNA-seq, Rai1, nicotinamide adenine dinucleotide, non-canonical initiating nucleotide, transcription, transcription initiation, transcription start site,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• endoribonukleasy metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• NAD metabolismus   MeSH
• nukleotidy genetika   MeSH
• počátek transkripce fyziologie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• RNA čepičky genetika   MeSH
• transkriptom genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• endoribonukleasy MeSH
• mRNA decapping enzymes MeSH Prohlížeč
• NAD MeSH
• nukleotidy MeSH
• RNA čepičky MeSH

Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m6A per RNA molecule. ONT sequencing predicted three m6A positions. Employing orthogonal SELECT method, we confirmed m6A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

• Klíčová slova
• 6-methyladenosine, LC-MS, m6A SELECT, RNA modification, Viroid, direct RNA-seq,
• MeSH
• konformace nukleové kyseliny MeSH
• kruhová RNA * genetika   metabolismus   MeSH
• nemoci rostlin virologie   MeSH
• posttranskripční úpravy RNA * MeSH
• replikace viru MeSH
• RNA virová * genetika   metabolismus   chemie   MeSH
• sekvenční analýza RNA MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kruhová RNA * MeSH
• RNA virová * MeSH

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.

• MeSH
• genetické vektory genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• RNA analýza   metabolismus   MeSH
• sekvence nukleotidů MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny vázající RNA MeSH
• RNA MeSH

In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.

• MeSH
• dimerizace MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.

• Klíčová slova
• RNA decay, cell biology, cell line, genetics, genomics, human, mouse, stress response,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   genetika   MeSH
• DNA-helikasy metabolismus   genetika   MeSH
• exoribonukleasy * metabolismus   genetika   MeSH
• fyziologický stres * genetika   MeSH
• lidé MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny vázající poly-ADP-ribosu * metabolismus   genetika   MeSH
• proteiny vázající RNA MeSH
• ribozomy metabolismus   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• RRM proteiny * metabolismus   genetika   MeSH
• sekvenční analýza RNA * MeSH
• stabilita RNA * genetika   MeSH
• stresová tělíska metabolismus   genetika   MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• DNA-helikasy MeSH
• exoribonukleasy * MeSH
• G3BP1 protein, human MeSH Prohlížeč
• G3BP2 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny vázající poly-ADP-ribosu * MeSH
• proteiny vázající RNA MeSH
• RNA-helikasy MeSH
• RRM proteiny * MeSH
• XRN1 protein, human MeSH Prohlížeč

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

• Klíčová slova
• Dosage compensation, Hrp48, RNA binding protein, RNA recognition motif, Translation regulation,
• MeSH
• 3' nepřekládaná oblast * MeSH
• DNA vazebné proteiny MeSH
• Drosophila melanogaster * metabolismus   genetika   MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• messenger RNA metabolismus   genetika   chemie   MeSH
• proteiny Drosophily * metabolismus   chemie   genetika   MeSH
• proteiny vázající RNA * metabolismus   chemie   genetika   MeSH
• proteosyntéza MeSH
• simulace molekulární dynamiky MeSH
• transkripční faktory metabolismus   chemie   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast * MeSH
• DNA vazebné proteiny MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• Hrb27C protein, Drosophila MeSH Prohlížeč
• messenger RNA MeSH
• msl-2 protein, Drosophila MeSH Prohlížeč
• proteiny Drosophily * MeSH
• proteiny vázající RNA * MeSH
• transkripční faktory MeSH

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

• MeSH
• buněčné linie MeSH
• G-kvadruplexy * MeSH
• Hepacivirus genetika   fyziologie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• replikace viru MeSH
• RNA virová biosyntéza   chemie   genetika   metabolismus   MeSH
• RNA-dependentní RNA-polymerasa metabolismus   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH

RNA interference (RNAi) has become an important tool to study and utilize gene silencing by introducing short interfering RNA (siRNA). In order to predict the most efficient siRNAs, a new software tool, RNA Workbench (RNAWB), has been designed and is freely available (after registration) on http://www.rnaworkbench.com. In addition to the standard selection rules, RNAWB includes the possibility of statistical analyses of the applied selection rules (criteria). The role of RNA secondary structures in the RNA interference process as well as the application of sequence rules are discussed to show the applicability of the software.

• MeSH
• algoritmy * MeSH
• genový targeting metody   MeSH
• malá interferující RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• RNA interference * MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza RNA metody   MeSH
• sekvenční seřazení metody   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH

Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.

• MeSH
• benchmarking MeSH
• cirkulující mikroRNA * genetika   MeSH
• lidé MeSH
• mikro RNA * genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cirkulující mikroRNA * MeSH
• mikro RNA * MeSH