cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.9699137
title: Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage

description: With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.
DOI: 10.6084/m9.figshare.9699137.v2
title: Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage

description: With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.
DOI: 10.6084/m9.figshare.9702449
title: ZFP217 regulates adipogenesis by controlling mitotic clonal expansion in a METTL3-m<sup>6</sup>A dependent manner

description: Obesity is becoming a global problem. Research into the detailed mechanism of adipocyte development is crucial for the treatment of excess fat. Zinc finger protein 217 plays roles in adipogenesis. However, the underlying mechanism remains unclear. Here, we demonstrated that ZFP217 knockdown prevented the mitotic clonal expansion process and caused adipogenesis inhibition. Depletion of ZFP217 increased the expression of the m<sup>6</sup>A methyltransferase METTL3, which upregulated the m<sup>6</sup>A level of cyclin D1 mRNA. METTL3 knockdown rescued the siZFP217-inhibited MCE and promoted CCND1 expression. YTH domain family 2 recognized and degraded the methylated CCND1 mRNA, leading to the downregulation of CCND1. Consequently, cell-cycle progression was blocked, and adipogenesis was inhibited. YTHDF2 knockdown relieved siZFP217-inhibited adipocyte differentiation. These findings reveal that ZFP217 knockdown–induced adipogenesis inhibition was caused by CCND1, which was mediated by METTL3 and YTHDF2 in an m<sup>6</sup>A-dependent manner. We have provided novel insight into the underlying molecular mechanisms by which m<sup>6</sup>A methylation is involved in the ZFP217 regulation of adipogenesis.
DOI: 10.6084/m9.figshare.9702449.v2
title: ZFP217 regulates adipogenesis by controlling mitotic clonal expansion in a METTL3-m<sup>6</sup>A dependent manner

description: Obesity is becoming a global problem. Research into the detailed mechanism of adipocyte development is crucial for the treatment of excess fat. Zinc finger protein 217 plays roles in adipogenesis. However, the underlying mechanism remains unclear. Here, we demonstrated that ZFP217 knockdown prevented the mitotic clonal expansion process and caused adipogenesis inhibition. Depletion of ZFP217 increased the expression of the m<sup>6</sup>A methyltransferase METTL3, which upregulated the m<sup>6</sup>A level of cyclin D1 mRNA. METTL3 knockdown rescued the siZFP217-inhibited MCE and promoted CCND1 expression. YTH domain family 2 recognized and degraded the methylated CCND1 mRNA, leading to the downregulation of CCND1. Consequently, cell-cycle progression was blocked, and adipogenesis was inhibited. YTHDF2 knockdown relieved siZFP217-inhibited adipocyte differentiation. These findings reveal that ZFP217 knockdown–induced adipogenesis inhibition was caused by CCND1, which was mediated by METTL3 and YTHDF2 in an m<sup>6</sup>A-dependent manner. We have provided novel insight into the underlying molecular mechanisms by which m<sup>6</sup>A methylation is involved in the ZFP217 regulation of adipogenesis.
DOI: 10.6084/m9.figshare.9684494
title: MicroRNA-142-3p suppresses endometriosis by regulating KLF9-mediated autophagy <i>in vitro</i> and <i>in vivo</i>

description: The detailed pathogenesis of endometriosis remains largely unclear despite decades of research. Recent studies have demonstrated that miRNAs plays an important role in endometriosis. The expression of miR-142-3p was decreased in ectopic endometrial tissues, while KLF9 and VEGFA expression levels were increased. Overexpression of miR-142-3p or knockdown of KLF9 significantly suppressed CRL-7566 cell proliferation and metastasis, induced cell apoptosis, and decreased both cell autophagy and vascularization. Additionally, KLF9 was confirmed to be a direct target of miR-142-3p and to directly bind to the promoter of the <i>VEGFA</i> gene, regulating its expression. Finally, intraperitoneal injection of miR-142-3p lentivirus significantly attenuated ectopic endometriotic lesions <i>in vivo</i>.miR-142-3p directly targeted KLF9, regulated VEGFA expression, and was protective against the growth of ectopic endometriotic lesions. Therefore, the miR-142-3p/KLF9/VEGFA signalling pathway may be a potential target in endometriosis treatment.
DOI: 10.6084/m9.figshare.9684494.v1
title: MicroRNA-142-3p suppresses endometriosis by regulating KLF9-mediated autophagy <i>in vitro</i> and <i>in vivo</i>

description: The detailed pathogenesis of endometriosis remains largely unclear despite decades of research. Recent studies have demonstrated that miRNAs plays an important role in endometriosis. The expression of miR-142-3p was decreased in ectopic endometrial tissues, while KLF9 and VEGFA expression levels were increased. Overexpression of miR-142-3p or knockdown of KLF9 significantly suppressed CRL-7566 cell proliferation and metastasis, induced cell apoptosis, and decreased both cell autophagy and vascularization. Additionally, KLF9 was confirmed to be a direct target of miR-142-3p and to directly bind to the promoter of the <i>VEGFA</i> gene, regulating its expression. Finally, intraperitoneal injection of miR-142-3p lentivirus significantly attenuated ectopic endometriotic lesions <i>in vivo</i>.miR-142-3p directly targeted KLF9, regulated VEGFA expression, and was protective against the growth of ectopic endometriotic lesions. Therefore, the miR-142-3p/KLF9/VEGFA signalling pathway may be a potential target in endometriosis treatment.
DOI: 10.6084/m9.figshare.9699137.v1
title: Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA splicing

description: With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA splicing play a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.
DOI: 10.6084/m9.figshare.9752456
title: Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA

description: During replication of long terminal repeat (LTR)-retrotransposons, their proteins and genome (g) RNA assemble into virus-like particles (VLPs) that are not infectious but functionally related to retroviral virions. Both virions and VLPs contain gRNA in a dimeric form, but contrary to retroviruses, little is known about how gRNA dimerization and packaging occurs in LTR-retrotransposons. The LTR-retrotransposon Ty1 from <i>Saccharomyces cerevisiae</i> is an informative model for studying LTR-retrotransposon and retrovirus replication. Using structural, mutational and functional analyses, we explored dimerization of Ty1 genomic RNA. We provide direct evidence that interactions of self-complementary PAL1 and PAL2 palindromic sequences localized within the 5′UTR are essential for Ty1 gRNA dimer formation. Mutations disrupting PAL1-PAL2 complementarity restricted RNA dimerization <i>in vitro</i> and Ty1 mobility <i>in vivo</i>. Although dimer formation and mobility of these mutants was inhibited, our work suggests that Ty1 RNA can dimerize via alternative contact points. In contrast to previous studies, we cannot confirm a role for PAL3, tRNA<sub>i</sub><sup>Met</sup> as well as recently proposed initial kissing-loop interactions in dimer formation. Our data also supports the critical role of Ty1 Gag in RNA dimerization. Mature Ty1 Gag binds in the proximity of sequences involved in RNA dimerization and tRNA<sub>i</sub><sup>Met</sup> annealing, but the 5′ pseudoknot in Ty1 RNA may constitute a preferred Gag-binding site. Taken together, these results expand our understanding of genome dimerization and packaging strategies utilized by LTR-retroelements.
DOI: 10.6084/m9.figshare.9752456.v1
title: Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA

description: During replication of long terminal repeat (LTR)-retrotransposons, their proteins and genome (g) RNA assemble into virus-like particles (VLPs) that are not infectious but functionally related to retroviral virions. Both virions and VLPs contain gRNA in a dimeric form, but contrary to retroviruses, little is known about how gRNA dimerization and packaging occurs in LTR-retrotransposons. The LTR-retrotransposon Ty1 from <i>Saccharomyces cerevisiae</i> is an informative model for studying LTR-retrotransposon and retrovirus replication. Using structural, mutational and functional analyses, we explored dimerization of Ty1 genomic RNA. We provide direct evidence that interactions of self-complementary PAL1 and PAL2 palindromic sequences localized within the 5′UTR are essential for Ty1 gRNA dimer formation. Mutations disrupting PAL1-PAL2 complementarity restricted RNA dimerization <i>in vitro</i> and Ty1 mobility <i>in vivo</i>. Although dimer formation and mobility of these mutants was inhibited, our work suggests that Ty1 RNA can dimerize via alternative contact points. In contrast to previous studies, we cannot confirm a role for PAL3, tRNA<sub>i</sub><sup>Met</sup> as well as recently proposed initial kissing-loop interactions in dimer formation. Our data also supports the critical role of Ty1 Gag in RNA dimerization. Mature Ty1 Gag binds in the proximity of sequences involved in RNA dimerization and tRNA<sub>i</sub><sup>Met</sup> annealing, but the 5′ pseudoknot in Ty1 RNA may constitute a preferred Gag-binding site. Taken together, these results expand our understanding of genome dimerization and packaging strategies utilized by LTR-retroelements.
DOI: 10.6084/m9.figshare.9702449.v1
title: ZFP217 regulates adipogenesis by controlling mitotic clonal expansion in a METTL3-m<sup>6</sup>A dependent manner

description: Obesity is becoming a global problem. Research into the detailed mechanism of adipocyte development is crucial for the treatment of excess fat. Zinc finger protein 217 plays roles in adipogenesis. However, the underlying mechanism remains unclear. Here, we demonstrated that ZFP217 knockdown prevented the mitotic clonal expansion process and caused adipogenesis inhibition. Depletion of ZFP217 increased the expression of the m<sup>6</sup>A methyltransferase METTL3, which upregulated the m<sup>6</sup>A level of cyclin D1 mRNA. METTL3 knockdown rescued the siZFP217-inhibited MCE and promoted CCND1 expression. YTH domain family 2 recognized and degraded the methylated CCND1 mRNA, leading to the downregulation of CCND1. Consequently, cell cycle progression was blocked, and adipogenesis was inhibited. YTHDF2 knockdown relieved siZFP217-inhibited adipocyte differentiation. These findings reveal that ZFP217 knockdown–induced adipogenesis inhibition was caused by CCND1, which was mediated by METTL3 and YTHDF2 in an m<sup>6</sup>A-dependent manner. We have provided novel insight into the underlying molecular mechanisms by which m<sup>6</sup>A methylation is involved in the ZFP217 regulation of adipogenesis.
DOI: 10.6084/m9.figshare.9724667.v1
title: Modulation of <i>PDCD1</i> exon 3 splicing

description: The <i>PDCD1</i> gene encodes PD-1, an important immune checkpoint protein and key immunotherapy target to treat cancer. <i>PDCD1</i> is alternatively spliced to generate an exon 3-skipped isoform PD-1Δ3 that has been suggested to play an antagonistic role to PD-1, but the mechanism underlying alternative splicing of <i>PDCD1</i> has never been explored. Here using a minigene system, we analysed the splicing pattern of <i>PDCD1</i> in multiple cell lines and confirmed exon 3 skipping as the main alternative splicing event. Using deletion analysis of exon 3, we mapped two splicing enhancers in the exon: ESE3a and ESE3b. Using mutagenesis, RNA-affinity chromatography, mass spectrometry as well as depletion and overexpression of MATR3, we defined MATR3 as a splicing activator during <i>PDCD1</i> exon 3 splicing that operates through binding to ESE3b. MATR3’s splicing-stimulatory activity is counteracted by an RNA secondary structure around ESE3b and an RNA helicase DDX5. Furthermore, we identified ASOs that efficiently promotes <i>PDCD1</i> exon 3 skipping in both minigene and endogenous-gene contexts. Our data support further study of the ASOs as potential drug candidates to treat cancer.
DOI: 10.6084/m9.figshare.9699227
title: The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds <i>in vivo</i>

description: Vascular endothelial growth factor <i>(VEGF)-A</i> is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-A<sub>xxx</sub> and anti-angiogenic, anti-permeability VEGF-A<sub>xxx</sub>b. <i>VEGF-A</i> splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of <i>VEGF-A in vivo</i>. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic <i>VEGF-A</i> terminal exon splicing (VEGF8ab) by insertion into the <i>ROSA26</i> genomic locus. dsRED expression denotes proximal splice site selection (VEGF-A<sub>xxx</sub>) and eGFP expression denotes distal splice site selection (VEGF-A<sub>xxx</sub>b). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous <i>VEGF-A</i> by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-A<sub>xxx</sub> and VEGF-A<sub>xxx</sub>b. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous <i>VEGF-A</i> splicing. The <i>VEGF-A</i> exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds <i>in vivo</i>
DOI: 10.6084/m9.figshare.9724667
title: Modulation of <i>PDCD1</i> exon 3 splicing

description: The <i>PDCD1</i> gene encodes PD-1, an important immune checkpoint protein and key immunotherapy target to treat cancer. <i>PDCD1</i> is alternatively spliced to generate an exon 3-skipped isoform PD-1Δ3 that has been suggested to play an antagonistic role to PD-1, but the mechanism underlying alternative splicing of <i>PDCD1</i> has never been explored. Here using a minigene system, we analysed the splicing pattern of <i>PDCD1</i> in multiple cell lines and confirmed exon 3 skipping as the main alternative splicing event. Using deletion analysis of exon 3, we mapped two splicing enhancers in the exon: ESE3a and ESE3b. Using mutagenesis, RNA-affinity chromatography, mass spectrometry as well as depletion and overexpression of MATR3, we defined MATR3 as a splicing activator during <i>PDCD1</i> exon 3 splicing that operates through binding to ESE3b. MATR3’s splicing-stimulatory activity is counteracted by an RNA secondary structure around ESE3b and an RNA helicase DDX5. Furthermore, we identified ASOs that efficiently promotes <i>PDCD1</i> exon 3 skipping in both minigene and endogenous-gene contexts. Our data support further study of the ASOs as potential drug candidates to treat cancer.
DOI: 10.6084/m9.figshare.9699227.v1
title: The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds <i>in vivo</i>

description: Vascular endothelial growth factor <i>(VEGF)-A</i> is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-A<sub>xxx</sub> and anti-angiogenic, anti-permeability VEGF-A<sub>xxx</sub>b. <i>VEGF-A</i> splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of <i>VEGF-A in vivo</i>. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic <i>VEGF-A</i> terminal exon splicing (VEGF8ab) by insertion into the <i>ROSA26</i> genomic locus. dsRED expression denotes proximal splice site selection (VEGF-A<sub>xxx</sub>) and eGFP expression denotes distal splice site selection (VEGF-A<sub>xxx</sub>b). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous <i>VEGF-A</i> by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-A<sub>xxx</sub> and VEGF-A<sub>xxx</sub>b. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous <i>VEGF-A</i> splicing. The <i>VEGF-A</i> exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds <i>in vivo</i>
DOI: 10.6084/m9.figshare.9771413
title: Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

description: Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.