7SK-controlled RNA polymerase II pausing orchestrates gene transcription with splicing
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ABSTRACT: RNA polymerase (Pol) II promoter-proximal pausing is a common regulatory step to initiate RNA synthesis and it is coordinated by a ribonucleoprotein complex scaffolded containing the noncoding RNA Rn7sk. However, how this transcriptional mechanism modulates gene expression programs in adult tissues is largely unknown. Here, we released paused RNA Pol II by depleting Rn7sk during mouse and human epidermal stem cell differentiation. Unexpectedly, the forced release of transcription robustly repressed RNA levels including numerous cell cycle regulators leading to cell cycle exit and differentiation. The repression of genes occurred in the absence of chromatin remodelling at promoters and enhancers. Instead, it was caused by splicing defects and co-transcriptional RNA degradation. However, rather than acting globally, Rn7sk modulated transcription gene-specifically. Genes that required Rn7sk for efficient transcription contained highly accessible promoters, lower guanosine and cytosine (GC) content, shorter introns, and weaker 3’ splice sites. In the absence of a tightly controlled Pol II pause release complex, these intrinsic gene properties cumulated in impaired splicing leading to RNA decay. Thus, 7SK-regulated transcriptional pausing is an essential step to coordinate transcription with splicing to permit productive transcription of a distinct set of genes.
Project description:RNA polymerase II promoter-proximal pausing is orchestrated by a ribonucleoprotein complex scaffolded by the noncoding RNA Rn7sk. However, how this interruption of transcription is mechanistically linked to RNA production remains largely unknown. Here, we show that forcing the pause release by germ-line deletion of Rn7sk was embryonic lethal, yet conditional deletion of Rn7sk enhanced stem cell differentiation in skin. To explore the immediate transcriptional mechanisms underpinning enhanced differentiation, we metabolically labelled newly-synthesized RNAs after Rn7sk deletion. Unexpectedly, forced pause release robustly repressed transcription specifically at cell cycle regulators, in the absence of chromatin remodeling at promoters and enhancers. Our results indicate that polymerase pausing affords the core elongation machinery time to properly assemble, and forced elongation triggers splicing defects and nuclear RNA decay. Cell cycle regulators appear highly sensitive to mis-regulation of the elongation machinery due to unique genomic features of high promoter accessibility and low GC–content in the gene body. Transcriptional pausing thus serves as a rate-limiting step in controlling cell division.
Project description:RNA polymerase II promoter-proximal pausing is orchestrated by a ribonucleoprotein complex scaffolded by the noncoding RNA Rn7sk. However, how this interruption of transcription is mechanistically linked to RNA production remains largely unknown. Here, we show that forcing the pause release by germ-line deletion of Rn7sk was embryonic lethal, yet conditional deletion of Rn7sk enhanced stem cell differentiation in skin. To explore the immediate transcriptional mechanisms underpinning enhanced differentiation, we metabolically labelled newly-synthesized RNAs after Rn7sk deletion. Unexpectedly, forced pause release robustly repressed transcription specifically at cell cycle regulators, in the absence of chromatin remodeling at promoters and enhancers. Our results indicate that polymerase pausing affords the core elongation machinery time to properly assemble, and forced elongation triggers splicing defects and nuclear RNA decay. Cell cycle regulators appear highly sensitive to mis-regulation of the elongation machinery due to unique genomic features of high promoter accessibility and low GC–content in the gene body. Transcriptional pausing thus serves as a rate-limiting step in controlling cell division.
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-chip data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis in human cells. Multiomics analysis reveals that U2 snRNP inhibition increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity in the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires formation of functional spliceosomes, and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.
Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.
Project description:Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.
Project description:The controlled release of promoter-proximal RNA polymerase II (Pol II) pausing into productive elongation is a major step in gene regulation. Yet, functionally analyzing Pol II pausing is difficult because the factors that regulate pause release are generally essential. Here, we identified heterozygous loss-of-function mutations in SUPT5H, encoding SPT5, in individuals with β-thalassemia trait unlinked to the β-globin locus. Recapitulating the pathogenic mutations in human erythroid cells or acute treatment with transcription inhibitors replicates the patient phenotype of altered globin gene expression. Furthermore, in SUPT5H-edited cells, Pol II pause release was globally disrupted. We observed dynamic patterns of pausing at cell cycle and erythroid differentiation genes at the transition from progenitors to precursors during erythropoiesis. At this same transition, in SUPT5H-edited cells, we observed delayed differentiation and altered cell cycle kinetics, as well as a delay in the onset of gene expression programs. Therefore, hindering pause release perturbs cell cycle and delays differentiation at key stages of erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.
Project description:The controlled release of promoter-proximal RNA polymerase II (Pol II) pausing into productive elongation is a major step in gene regulation. Yet, functionally analyzing Pol II pausing is difficult because the factors that regulate pause release are generally essential. Here, we identified heterozygous loss-of-function mutations in SUPT5H, encoding SPT5, in individuals with β-thalassemia trait unlinked to the β-globin locus. Recapitulating the pathogenic mutations in human erythroid cells or acute treatment with transcription inhibitors replicates the patient phenotype of altered globin gene expression. Furthermore, in SUPT5H-edited cells, Pol II pause release was globally disrupted. We observed dynamic patterns of pausing at cell cycle and erythroid differentiation genes at the transition from progenitors to precursors during erythropoiesis. At this same transition, in SUPT5H-edited cells, we observed delayed differentiation and altered cell cycle kinetics, as well as a delay in the onset of gene expression programs. Therefore, hindering pause release perturbs cell cycle and delays differentiation at key stages of erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.