Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA280492 | ENA |
REPOSITORIES: ENA
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Project description:The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. two treatements and one control
2015-04-07 | E-GEOD-67617 | biostudies-arrayexpress
Project description:The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.
2015-04-07 | GSE67617 | GEO
Project description:Transcriptome analysis of total RNA samples from heart tissue of knockout mice Alternative splicing is the main mechanism to increase protein diversity from an mRNA. Heterogeneous ribonucleoprotein (hnRNP) family members are vital regulators of alternative splicing. The hnRNP A1 is the most well-known protein in this family, but its role in embryonic development is not well understood. We generated hnRNP A1 knockout mice to study the function of hnRNP A1 in vivo. The hnRNP A1-depleted mice showed embryonic lethality because of muscle developmental defects. In a previous study, cellular hnRNP A2/B1 was reported to be capable of compensating for the expression of hnRNP A1. However, this phenomenon did not occur in the hnRNP A1 heterozygous mice in vivo. We demonstrated that hnRNP A1 regulated muscle-related genes expression and alternative splicing. In summary, our data demonstrated that hnRNP A1 plays a critical role in embryonic muscle development. Understanding the effects of hnRNP A1 in vivo may help to define the function of hnRNP A1 in alternative splicing.
2016-12-05 | GSE79076 | GEO
Project description:We used NEBNext Ultra Directional RNA Library Prep Kits to prepare RNA-seq libraries of total RNA from hnRNP A2/B1 and A1 depleted A549 cells. Pro-seq libraries were prepared from A549 cells using Illumina adapters hnRNP A2/B1 and A1 depleted A549 cells were generated by lentiviral infections of shRNA constructs. RNAs were isolated using Trizol.
2016-05-17 | E-GEOD-76151 | biostudies-arrayexpress
Project description:Transcriptome profiling of hnRNP A2/B1 and A1 depleted cells
| PRJNA306484 | ENA
Project description:Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. RNAseq for control, hnRNP A1, hnRNP A2/B1, hnRNP H1, hnRNP F, hnRNP M, and hnRNP U siRNA treated human 293T cells
2012-02-20 | E-GEOD-34995 | biostudies-arrayexpress
Project description:Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. CLIPseq for hnRNP A1, hnRNP A2/B1, hnRNP F, hnRNP M, and hnRNP U in human 293T cells
2012-02-20 | E-GEOD-34993 | biostudies-arrayexpress
Project description:Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. In triplicate, polyA-selected RNA was extracted from control, hnRNP A1, hnRNP A2/B1, hnRNP H1, hnRNP F, hnRNP M, and hnRNP U siRNA treated human 293T cells, and hybridized to custom splice-junction arrays
2012-02-20 | E-GEOD-34992 | biostudies-arrayexpress
Project description:Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicative of their concerted function in mRNA metabolism.
2013-11-01 | GSE49225 | GEO
Project description:Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicative of their concerted function in mRNA metabolism. Ribonucleoprotein Immunoprecipitation (RIP) using hnRNP A1 specific antibody was performed in nuclear extracts from HuR WT and HuR KO Mouse Embryonic Fibroblasts (MEFs). RNA isolated from these IPs together with nuclear RNA from the two cell types, was subjected to microarray analysis. Three biological replicates, representing three independent experiments, are available for each condition except in the case of nuclear RNA isolated from HuR WT MEFs that one replicate didn’t pass the quality control.
2013-11-01 | E-GEOD-49225 | biostudies-arrayexpress