cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.13385284.v1
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.
DOI: 10.6084/m9.figshare.13513478
title: Dissecting protein domain variability in the core RNA interference machinery of five insect orders

description: RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (<i>K</i>) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.
DOI: 10.6084/m9.figshare.13526192
title: Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing

description: Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50–300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about ~1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the <i>POLR3E</i> locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analysing the roles of msRNAs in cells, development and diseases.
DOI: 10.6084/m9.figshare.13513478.v1
title: Dissecting protein domain variability in the core rna interference machinery of five insect orders

description: RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (<i>K</i>) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.
DOI: 10.6084/m9.figshare.13513478.v2
title: Dissecting protein domain variability in the core RNA interference machinery of five insect orders

description: RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (<i>K</i>) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.
DOI: 10.6084/m9.figshare.13621745
title: Ribosome profiling analysis of human skeletal muscle identifies reduced translation of mitochondrial proteins with age

description: With advancing age, human muscle loses strength and function, but the molecular causes of these losses are unknown. Skeletal muscle shows an age-dependent decline in the levels of different proteins, but whether such decline is associated with reduced translation has not been studied. To address this gap of knowledge, we used the technique of ribosome profiling to study translation in muscle from middle-aged and old individuals. Using ribosome occupancy as a measure of translation status, several mRNAs showed differential translation with age. Older age was associated with lower translation of myosin and titin isoforms and more broadly with the translation of proteins involved in oxidative phosphorylation encoded by the mitochondrial genome. Based on our findings, we propose that mitochondrial proteins are less translated in old skeletal muscle.
DOI: 10.6084/m9.figshare.13621745.v1
title: Ribosome profiling analysis of human skeletal muscle identifies reduced translation of mitochondrial proteins with age

description: With advancing age, human muscle loses strength and function, but the molecular causes of these losses are unknown. Skeletal muscle shows an age-dependent decline in the levels of different proteins, but whether such decline is associated with reduced translation has not been studied. To address this gap of knowledge, we used the technique of ribosome profiling to study translation in muscle from middle-aged and old individuals. Using ribosome occupancy as a measure of translation status, several mRNAs showed differential translation with age. Older age was associated with lower translation of myosin and titin isoforms and more broadly with the translation of proteins involved in oxidative phosphorylation encoded by the mitochondrial genome. Based on our findings, we propose that mitochondrial proteins are less translated in old skeletal muscle.
DOI: 10.6084/m9.figshare.13526192.v1
title: Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing.

description: Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50-300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about ~1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the <i>POLR3E</i> locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analyzing the roles of msRNAs in cells, development and diseases.
DOI: 10.6084/m9.figshare.13487145
title: Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach

description: Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs’ population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs’ function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.
DOI: 10.6084/m9.figshare.13487145.v1
title: Discovery and Functional Understanding of MiRNAs in Mollusks: A Genome-wide Profiling Approach

description: Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in mollusks. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of mollusks through genome scanning. Afterward, the miRNAs’ population, isoforms, organization, and function were characterised in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs’ function in biomineralization, immune and stress response, as well as growth and development in mollusks. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in mollusks, as well as other invertebrates.
DOI: 10.6084/m9.figshare.13487145.v2
title: Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach

description: Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs’ population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs’ function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.
DOI: 10.6084/m9.figshare.14052264
title: CBRPP: a new RNA-centric method to study RNA–protein interactions

description: RNA and protein are interconnected biomolecules that can influence each other’s life cycles and functions through physical interactions. Abnormal RNA–protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA–protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA–protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA–protein complexes manipulation <i>in vitro</i>. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.
DOI: 10.6084/m9.figshare.14052264.v1
title: CBRPP: a new RNA-centric method to study RNA–protein interactions

description: RNA and protein are interconnected biomolecules that can influence each other’s life cycles and functions through physical interactions. Abnormal RNA–protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA–protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA–protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA–protein complexes manipulation <i>in vitro</i>. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.
DOI: 10.6084/m9.figshare.13513481
title: Bulges control pri-miRNA processing in a position and strand-dependent manner

description: MicroRNAs (miRNAs) play critical roles in gene expression and numerous human diseases. The success of miRNA biogenesis is largely determined by the primary miRNA (pri-miRNA) processing by the DROSHA-DGCR8 complex, called Microprocessor. Here, we analysed the high-throughput pri-miRNA processing assays and secondary structures of pri-miRNAs to investigate the roles of bulges in the pri-miRNA processing. We found that bulges in multiple places control both the cleavage efficiency and accuracy of pri-miRNA processing. These bulges were shown to act on Microprocessor via its catalytic subunit, DROSHA, and function in a position and strand-dependent manner. Interestingly, we discovered that the enriched and conserved bulges, called midB, can correct DROSHA orientation on pri-miRNAs, thereby enhancing production of miRNAs. The revealed functions of the bulges help improve our understanding of pri-miRNA processing and suggest their potential roles in miRNA biogenesis regulation.
DOI: 10.6084/m9.figshare.13513481.v1
title: Bulges control pri-miRNA processing in a position and strand-dependent manner

description: MicroRNAs (miRNAs) play critical roles in gene expression and numerous human diseases. The success of miRNA biogenesis is largely determined by the primary miRNA (pri-miRNA) processing by the DROSHA-DGCR8 complex, called Microprocessor. Here, we analysed the high-throughput pri-miRNA processing assays and secondary structures of pri-miRNAs to investigate the roles of bulges in the pri-miRNA processing. We found that bulges in multiple places control both the cleavage efficiency and accuracy of pri-miRNA processing. These bulges were shown to act on Microprocessor via its catalytic subunit, DROSHA, and function in a position and strand-dependent manner. Interestingly, we discovered that the enriched and conserved bulges, called midB, can correct DROSHA orientation on pri-miRNAs, thereby enhancing production of miRNAs. The revealed functions of the bulges help improve our understanding of pri-miRNA processing and suggest their potential roles in miRNA biogenesis regulation.
DOI: 10.6084/m9.figshare.13670006
title: Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

description: While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, <i>Haloferax volc
anii</i>, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in <i>H. volcanii</i>, named <b>S</b>mall RNA in <b>H</b>aloferax <b>Oxi</b>dative Stress (<b>SHOxi</b>). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative ‘seed’ region within the predicted stem loop of SHOxi. Malic enzyme catalyzes the oxidative decarboxylation of malate into pyruvate using NAD<sup>+</sup> as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD<sup>+</sup>/NADH ratio, resulting from the direct RNA–RNA interaction between SHOxi and its trans-target was essential for the survival of <i>H. volcanii</i> to oxidative stress. These findings indicate that SHOxi likely regulates redox homoeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.
DOI: 10.6084/m9.figshare.13338398
title: Towards the mechanism(s) of YB-3 synthesis regulation by YB-1

description: Y-box binding proteins are members of the family of proteins containing the evolutionarily conserved cold shock domain. Their cellular functions are quite diverse, including transcription and translation regulation, participation in pre-mRNA splicing, mRNA stabilization and packaging into mRNPs, involvement in DNA repair, and some others. To date, we know little about the plausible functional interchangeability of Y-box binding proteins. Our previous finding was that in YB-1-null HEK293T cells the synthesis of YB-3 is enhanced, thus enabling YB-3 to interact with a larger set of mRNAs and compensate for the YB-1 absence. We suggested the existence of a mechanism of YB-3 synthesis regulation by its paralog, YB-1. Here we demonstrate that YB-1 participates in the translational control and stabilization of <i>YB-3</i> mRNA through untranslated regions of <i>YB-3</i> mRNA.