cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.14114926
title: Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen <i>Clostridioides difficile</i>

description: Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen <i>Clostridioides (Clostridium) difficile</i>. Some of them are <i>trans</i>-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in <i>C. difficile</i> with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with <i>C. difficile</i> Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in <i>C. difficile</i>. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to <i>trans</i>-encoded RNAs, new categories of Hfq ligands were found including <i>cis</i>-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in <i>C. difficile</i>. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.
DOI: 10.6084/m9.figshare.13521484
title: Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA

description: RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with &gt;80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or &gt;26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.
DOI: 10.6084/m9.figshare.13526195.v1
title: A chromatin-associated splicing isoform of <i>OIP5-AS1</i> acts in <i>cis</i> to regulate the <i>OIP5</i> oncogene

description: A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. <i>OIP5-AS1</i> (also known as <i>Cyrano</i>), a poorly characterized lncRNA expressed antisense to the <i>OIP5</i> oncogene, is deregulated in multiple cancers. We showed that one of the <i>OIP5-AS1</i> splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, <i>OIP5</i>, strongly suggesting a functional coupling between <i>OIP5</i> and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the <i>OIP5</i> oncogene.
DOI: 10.6084/m9.figshare.13943290
title: Direct RNA–RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention

description: Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3ʹUTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA–RNA interactions. Using an RNA pull-down procedure adapted to select only RNAs engaged in direct RNA–RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5ʹ end of Neat1 demonstrating that they are located in the shell region of paraspeckles.
DOI: 10.6084/m9.figshare.13378777.v1
title: Hormonal regulation of microRNA expression dynamics in the gut of the yellow fever mosquito <i>Aedes aegypti</i>

description: The yellow fever mosquito <i>Aedes aegypti</i> is an obligatory blood feeder and a major arboviral disease vector, evoking severe public health concerns worldwide. In adult female mosquitoes, the gut is critical for blood digestion and pathogen entry. We aimed for a systematic exploration of microRNA expression dynamics in the gut during the gonadotrophic cycle. Small RNA libraries were constructed from female mosquito gut tissues at five time points. Unsupervised hierarchical clustering revealed three expression clusters (early, mid and late) peaking at sequential time points – 24, 48 and 72 h posteclosion. Differentially expressed miRNAs were identified at 24 h post-blood meal (PBM). Depletions of Methoprene-tolerant [Met; the juvenile hormone (JH) receptor] and Ecdysone receptor [EcR; the receptor to 20-hydroxyecdysone (20E)] were performed using dsRNA to these genes to investigate impacts on microRNA expressions. Our results suggest that Met-mediated signalling downregulates miRNA expression from the early cluster and upregulates that from the late cluster. EcR signalling either up- or downregulated miRNA levels at 24 h PBM, indicating a differential effect of this receptor in miRNA gene expression. Furthermore, miR-281, which is the most abundant miRNA in the gut tissue, is induced and repressed by Met- and EcR-mediated signalling, respectively. Systematic depletion using synthetic antagomir and phenotype examinations indicate that miR-281 is obligatory for the normal progression of blood digestion, ovarian development and reproduction. Collectively, this study unveils expression dynamics of microRNAs in the female gut tissue during the gonadotrophic cycle and demonstrates that they are affected by JH and 20E signalling.
DOI: 10.6084/m9.figshare.13553724
title: Characterization of synthetic riboswitch in cell-free protein expression systems

description: Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5ʹ-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in <i>E. coli</i> cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3ʹ-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both <i>E. coli</i> extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors – a reduced fraction of mRNA in the ‘ON’ state and a considerably lower translation initiation rate in the riboswitch – contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.
DOI: 10.6084/m9.figshare.14052298
title: Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants

description: In all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2ʹ-<i>O</i>-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB). Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2ʹ-<i>O</i>-methylation sites in <i>A. thaliana</i> Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2ʹ-<i>O</i>-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2ʹ-<i>O</i>-methylation at specific rRNA sites suggesting functional/structural interconnections of 2ʹ-<i>O</i>-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plants.
DOI: 10.6084/m9.figshare.14114926.v1
title: Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen <i>Clostridioides difficile</i>

description: Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen <i>Clostridioides (Clostridium) difficile</i>. Some of them are <i>trans</i>-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in <i>C. difficile</i> with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with <i>C. difficile</i> Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in <i>C. difficile</i>. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to <i>trans</i>-encoded RNAs, new categories of Hfq ligands were found including <i>cis</i>-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in <i>C. difficile</i>. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.
DOI: 10.6084/m9.figshare.13521484.v1
title: Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA

description: RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with &gt;80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or &gt;26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.
DOI: 10.6084/m9.figshare.13943290.v2
title: Direct RNA–RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention

description: Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3ʹUTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA–RNA interactions. Using an RNA pull-down procedure adapted to select only RNAs engaged in direct RNA–RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5ʹ end of Neat1 demonstrating that they are located in the shell region of paraspeckles.
DOI: 10.6084/m9.figshare.13553724.v1
title: Characterization of synthetic riboswitch in cell-free protein expression systems

description: Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5ʹ-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in <i>E. coli</i> cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3ʹ-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both <i>E. coli</i> extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors – a reduced fraction of mRNA in the ‘ON’ state and a considerably lower translation initiation rate in the riboswitch – contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.
DOI: 10.6084/m9.figshare.13943290.v1
title: Direct RNA-RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention

description: Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3ʹUTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA-RNA interactions. Using a RNA pull-down procedure adapted to select only RNAs engaged in direct RNA-RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5ʹ end of Neat1 demonstrating that they are located in the shell region of paraspeckles.
DOI: 10.6084/m9.figshare.14052298.v1
title: Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants

description: In all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2ʹ-<i>O</i>-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB). Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2ʹ-<i>O</i>-methylation sites in <i>A. thaliana</i> Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2ʹ-<i>O</i>-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2ʹ-<i>O</i>-methylation at specific rRNA sites suggesting functional/structural interconnections of 2ʹ-<i>O</i>-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plants.
DOI: 10.6084/m9.figshare.13385284
title: Polynucleotide phosphorylase and RNA helicase CshA cooperate in <i>Bacillus subtilis</i> mRNA decay

description: Polynucleotide phosphorylase (PNPase), a 3ʹ exoribonuclease that degrades RNA in the 3ʹ-to-5ʹ direction, is the major mRNA decay activity in <i>Bacillus subtilis</i>. PNPase is known to be inhibited <i>in vitro</i> by strong RNA secondary structure, and rapid mRNA turnover <i>in vivo</i> is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in <i>B. subtilis</i> is CshA. We found for three small, monocistronic mRNAs that, for some RNA sequences, PNPase processivity was unimpeded even without CshA, whereas others required CshA for efficient degradation. A novel colour screen for decay of mRNA in <i>B. subtilis</i> was created, using mRNA encoded by the <i>slrA</i> gene, which is degraded from its 3ʹ end by PNPase. A significant correlation between the predicted strength of a stem-loop structure, located in the body of the message, and PNPase processivity was observed. Northern blot analysis confirmed that PNPase processivity was greatly hindered by the internal RNA structure, and even more so in the absence of CshA. Three other <i>B. subtilis</i> RNA helicases did not appear to be involved in mRNA decay during vegetative growth. The results confirm the hypothesis that efficient 3ʹ exonucleolytic decay of <i>B. subtilis</i> RNA depends on the combined activity of PNPase and CshA.