cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.11474295.v1
title: Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner

description: Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5ʹ tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5ʹ tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.
DOI: 10.6084/m9.figshare.11919768
title: Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs

description: Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found <i>in vivo</i>. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNA<sup>Ala</sup> and tiRNA<sup>Gly</sup>. We show that natural 5ʹ-tiRNA<sup>Ala</sup> molecules assemble into G-quadruplex structures, and endogenous 5ʹ-tiRNA<sup>Gly</sup> possesses translation inhibition activity.
DOI: 10.6084/m9.figshare.11919768.v1
title: Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs

description: Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found <i>in vivo</i>. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNA<sup>Ala</sup> and tiRNA<sup>Gly</sup>. We show that natural 5ʹ-tiRNA<sup>Ala</sup> molecules assemble into G-quadruplex structures, and endogenous 5ʹ-tiRNA<sup>Gly</sup> possesses translation inhibition activity.
DOI: 10.6084/m9.figshare.11936397
title: LncRNAs in molluscan and mammalian stages of parasitic schistosomes are developmentally-regulated and coordinately expressed with protein-coding genes

description: Despite the low level expression of some long noncoding RNAs (lncRNAs), the differential expression of specific lncRNAs plays important roles during the development of many organisms. Schistosomes, parasitic flatworms that are responsible for schistosomiasis, infects over 200 million people resulting in chronic disease and hundreds of thousands of deaths. Schistosomes have a complex life cycle that transitions between molluscan and mammalian hosts. In a molluscan snail host, the sporocyst stage develops over 5 weeks undergoing asexual reproduction to give rise to free-swimming and infectious cercariae that penetrate human skin and eventually mature into egg producing worms in mammals. The tight integration of the sporocyst to the snail host hepatopancreas hinders the -omics study in the molluscan stage, so the sporocyst transcriptome has only been examined for lncRNAs in immature <i>in vitro</i> samples. Here we analyzed the <i>in vivo</i> mature sporocyst transcriptome to identify 4,930 total lncRNAs between the molluscan and mammalian stages of the parasite. We further demonstrate that the lncRNAs are differentially expressed in a development-dependent manner. In addition, we constructed a co-expression correlation network between lncRNAs and protein-coding (PC) genes that was used to identify clusters of lncRNA transcripts with potential functional relevance. We also describe lncRNA–lncRNA and lncRNA–kinome correlations that identify lncRNAs with prospective roles in gene regulation. Finally, our results show clear differential expression patterns of lncRNAs in host-dependent development stages of <i>S. mansoni</i> and ascribe potential functional roles in development based on predicted intracellular interaction.
DOI: 10.6084/m9.figshare.11936397.v1
title: LncRNAs in molluscan and mammalian stages of parasitic schistosomes are developmentally-regulated and coordinately expressed with protein-coding genes

description: Despite the low level expression of some long noncoding RNAs (lncRNAs), the differential expression of specific lncRNAs plays important roles during the development of many organisms. Schistosomes, parasitic flatworms that are responsible for schistosomiasis, infects over 200 million people resulting in chronic disease and hundreds of thousands of deaths. Schistosomes have a complex life cycle that transitions between molluscan and mammalian hosts. In a molluscan snail host, the sporocyst stage develops over 5 weeks undergoing asexual reproduction to give rise to free-swimming and infectious cercariae that penetrate human skin and eventually mature into egg producing worms in mammals. The tight integration of the sporocyst to the snail host hepatopancreas hinders the -omics study in the molluscan stage, so the sporocyst transcriptome has only been examined for lncRNAs in immature <i>in vitro</i> samples. Here we analyzed the <i>in vivo</i> mature sporocyst transcriptome to identify 4,930 total lncRNAs between the molluscan and mammalian stages of the parasite. We further demonstrate that the lncRNAs are differentially expressed in a development-dependent manner. In addition, we constructed a co-expression correlation network between lncRNAs and protein-coding (PC) genes that was used to identify clusters of lncRNA transcripts with potential functional relevance. We also describe lncRNA–lncRNA and lncRNA–kinome correlations that identify lncRNAs with prospective roles in gene regulation. Finally, our results show clear differential expression patterns of lncRNAs in host-dependent development stages of <i>S. mansoni</i> and ascribe potential functional roles in development based on predicted intracellular interaction.
DOI: 10.6084/m9.figshare.11919756
title: The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation

description: Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5ʹUTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.
DOI: 10.6084/m9.figshare.11958795
title: The core spliceosomal factor U2AF1 controls cell-fate determination via the modulation of transcriptional networks

description: Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.
DOI: 10.6084/m9.figshare.11871159
title: Study of key RNA metabolism proteins in <i>Enterococcus faecalis</i>

description: The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like <i>Enterococcus faecalis</i>. This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated <i>in vitro</i>, and the results are in accordance with a degradosome structure close to the one of <i>Bacillus subtilis</i>. At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the <i>Galleria mellonella</i> infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the Δ<i>cshA</i> mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5ʹUnstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.
DOI: 10.6084/m9.figshare.11871159.v1
title: Study of key RNA metabolism proteins in <i>Enterococcus faecalis</i>

description: The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like <i>Enterococcus faecalis</i>. This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated <i>in vitro</i>, and the results are in accordance with a degradosome structure close to the one of <i>Bacillus subtilis</i>. At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the <i>Galleria mellonella</i> infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the Δ<i>cshA</i> mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5ʹUnstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.
DOI: 10.6084/m9.figshare.11958795.v1
title: The core spliceosomal factor U2AF1 controls cell-fate determination via the modulation of transcriptional networks

description: Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.
DOI: 10.6084/m9.figshare.12851873
title: Coordination of transcriptional and translational regulations in human epithelial cells infected by <i>Listeria monocytogenes</i>

description: The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a time-course of infection of human intestinal epithelial cells by an epidemic strain of <i>Listeria monocytogenes</i>, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5ʹ-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (<i>PABPC1</i>) slowed down the intracellular multiplication of <i>Listeria monocytogenes</i>, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.
DOI: 10.6084/m9.figshare.12851873.v2
title: Coordination of transcriptional and translational regulations in human epithelial cells infected by <i>Listeria monocytogenes</i>

description: The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a time-course of infection of human intestinal epithelial cells by an epidemic strain of <i>Listeria monocytogenes</i>, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5ʹ-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (<i>PABPC1</i>) slowed down the intracellular multiplication of <i>Listeria monocytogenes</i>, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.
DOI: 10.6084/m9.figshare.12851873.v1
title: Coordination of transcriptional and translational regulations in human epithelial cells infected by <i>Listeria monocytogenes</i>

description: The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a time-course of infection of human intestinal epithelial cells by an epidemic strain of <i>Listeria monocytogenes</i>, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5ʹ-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (<i>PABPC1</i>) slowed down the intracellular multiplication of <i>Listeria monocytogenes</i>, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.
DOI: 10.6084/m9.figshare.12851849.v1
title: Competitive learning suggests circulating miRNA profiles for cancers decades prior to diagnosis

description: MicroRNAs are regulators of gene expressionand may be key markers in liquid biopsy.Early diagnosis is an effective means to increase patients’ overall survival. We generated genome-wide miRNA profiles from serum of patients and controls from the population-based Janus Serum Bank (JSB) and analysed them by bioinformatics and artificial intelligence approaches. JSB contains sera from 318,628 originally healthy persons, more than 96,000 of whom developed cancer. We selected 210 serum samples from patients with lung, colon or breast cancer at three time points prior to diagnosis (up to 32 years prior to diagnosis with median 5 years interval between TPs), one time-point after diagnosis and from individually matched controls. The controls were matched on age and year of all pre-diagnostic sampling time-points for the corresponding case. Using ANOVA we report 70 significantly deregulated markers (adjusted p-value&lt;0.05). The driver for the significance was the diagnostic time point (miR-575, miR-6821-5p, miR-630 with adjusted p-values&lt;10<sup>−10</sup>). Further, 91miRNAs were differently expressed in pre-diagnostic samples as compared to controls (nominal p
DOI: 10.6084/m9.figshare.12851864.v1
title: Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA

description: Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from <i>Deltaproteobacteria</i> and PlmCas12e from <i>Planctomycetes</i> can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22<sup>nd</sup> position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.