cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.19245933.v2
title: A novel role for nucleolin in splice site selection

description: Latent 5ʹ splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of initiator-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.
DOI: 10.6084/m9.figshare.19245933.v1
title: A novel role for nucleolin in splice site selection

description: Latent 5ʹ splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.
DOI: 10.6084/m9.figshare.19688962
title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

description: It has recently been shown that CFIm25, a canonical mRNA 3’ processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor <i>rex1</i>. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3’ processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25ʹs modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3’ processing factor.
DOI: 10.6084/m9.figshare.19688962.v2
title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

description: It has recently been shown that CFIm25, a canonical mRNA 3’ processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor <i>rex1</i>. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3’ processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25ʹs modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3’ processing factor.
DOI: 10.6084/m9.figshare.18865636
title: Genomically Hardwired Regulation of Gene Activity Orchestrates Cellular Iron Homeostasis in Arabidopsis

description: Iron (Fe) is an essential micronutrient which plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homoeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of <i>Arabidopsis thaliana</i> plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.
DOI: 10.6084/m9.figshare.18865636.v1
title: Genomically Hardwired Regulation of Gene Activity Orchestrates Cellular Iron Homeostasis in Arabidopsis

description: Iron (Fe) is an essential micronutrient which plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homoeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of <i>Arabidopsis thaliana</i> plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.
DOI: 10.6084/m9.figshare.19688962.v1
title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

description: It has recently been shown that CFIm25, a canonical mRNA 3’ processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor <i>rex1</i>. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3’ processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25ʹs modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3’ processing factor.
DOI: 10.6084/m9.figshare.20137232
title: Poly(I:C) transfection induces a pro-inflammatory cascade in murine mammary carcinoma and fibrosarcoma cells

description: Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.
DOI: 10.6084/m9.figshare.20137232.v1
title: Poly(I:C) transfection induces a pro-inflammatory cascade in murine mammary carcinoma and fibrosarcoma cells

description: Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.
DOI: 10.6084/m9.figshare.21546116
title: High-throughput mapping of RNA solvent accessibility at the single-nucleotide resolution by RtcB ligation between a fixed 5′-OH-end linker and unique 3′-P-end fragments from hydroxyl radical cleavage

description: Given the challenges for the experimental determination of RNA tertiary structures, probing solvent accessibility has become increasingly important to gain functional insights. Among various chemical probes developed, backbone-cleaving hydroxyl radical is the only one that can provide unbiased detection of all accessible nucleotides. However, the readouts have been based on reverse transcription (RT) stop at the cleaving sites, which are prone to false positives due to PCR amplification bias, early drop-off of reverse transcriptase, and the use of random primers in RT reaction. Here, we introduced a fixed-primer method called RL-Seq by performing RtcB Ligation (RL) between a fixed 5′-OH-end linker and unique 3′-P-end fragments from hydroxyl radical cleavage prior to high-throughput sequencing. The application of this method to <i>E. coli</i> ribosomes confirmed its ability to accurately probe solvent accessibility with high sensitivity (low required sequencing depth) and accuracy (strong correlation to structure-derived values) at the single-nucleotide resolution. Moreover, a near-perfect correlation was found between the experiments with and without using unique molecular identifiers, indicating negligible PCR biases in RL-Seq. Further improvement of RL-Seq and its potential transcriptome-wide applications are discussed.
DOI: 10.6084/m9.figshare.21546116.v1
title: High-throughput mapping of RNA solvent accessibility at the single-nucleotide resolution by RtcB ligation between a fixed 5′-OH-end linker and unique 3′-P-end fragments from hydroxyl radical cleavage

description: Given the challenges for the experimental determination of RNA tertiary structures, probing solvent accessibility has become increasingly important to gain functional insights. Among various chemical probes developed, backbone-cleaving hydroxyl radical is the only one that can provide unbiased detection of all accessible nucleotides. However, the readouts have been based on reverse transcription (RT) stop at the cleaving sites, which are prone to false positives due to PCR amplification bias, early drop-off of reverse transcriptase, and the use of random primers in RT reaction. Here, we introduced a fixed-primer method called RL-Seq by performing RtcB Ligation (RL) between a fixed 5′-OH-end linker and unique 3′-P-end fragments from hydroxyl radical cleavage prior to high-throughput sequencing. The application of this method to <i>E. coli</i> ribosomes confirmed its ability to accurately probe solvent accessibility with high sensitivity (low required sequencing depth) and accuracy (strong correlation to structure-derived values) at the single-nucleotide resolution. Moreover, a near-perfect correlation was found between the experiments with and without using unique molecular identifiers, indicating negligible PCR biases in RL-Seq. Further improvement of RL-Seq and its potential transcriptome-wide applications are discussed.
DOI: 10.6084/m9.figshare.19388481
title: Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection

description: Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus <i>Magnaporthe oryzae</i>, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-<i>M. oryzae</i> interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.
DOI: 10.6084/m9.figshare.19388481.v1
title: Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection

description: Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus <i>Magnaporthe oryzae</i>, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-<i>M. oryzae</i> interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.
DOI: 10.6084/m9.figshare.19388481.v2
title: Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection

description: Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus <i>Magnaporthe oryzae</i>, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-<i>M. oryzae</i> interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.
DOI: 10.6084/m9.figshare.20310627
title: Investigating the role of RNA structures in transcriptional pausing using in vitro assays and in silico analyses

description: Transcriptional pausing occurs across the bacterial genome but the importance of this mechanism is still poorly understood. Only few pauses were observed during the previous decades, leaving an important gap in understanding transcription mechanisms. Using the well-known <i>Escherichia coli hisL</i> and <i>trpL</i> pause sites as models, we describe here the relation of pause sites with upstream RNA structures suspected to stabilize pausing. We find that the transcription factor NusA influences the pause half-life at <i>leuL, pheL</i> and <i>thrL</i> pause sites. Using a mutagenesis approach, we observe that transcriptional pausing is affected in all tested pause sites, suggesting that the upstream RNA sequence is important for transcriptional pausing. Compensatory mutations assessing the presence of RNA hairpins did not yield clear conclusions, indicating that complex RNA structures or transcriptional features may be playing a role in pausing. Moreover, using a bioinformatic approach, we explored the relation between a DNA consensus sequence important for pausing and putative hairpins among thousands of pause sites in <i>E. coli</i>. We identified 2125 sites presenting hairpin-dependent transcriptional pausing without consensus sequence, suggesting that this mechanism is widespread across <i>E. coli</i>. This study paves the way to understand the role of RNA structures in transcriptional pausing.