Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1089218 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28380354_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380355_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380356_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380357_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380358_subreads.fastq.gz | Fastqsanger.gz |
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Project description:Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.
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Project description:Ribosomal DNA (rDNA) repeats harbor ribosomal RNA (rRNA) genes and intergenic spacers (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding rRNA components of ribosomes. IGS-associated Pol II prevents Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli and rRNA production. Here, compartment-enriched proximity-dependent biotin identification (compBioID) revealed the TATA-less-promoter-binding TBPL1 and transcription-regulatory PAF1 with nucleolar Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I-associated ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the nucleolar interactome of Pol II and show that its regulation by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and function.
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Project description:Gene expression in metazoans is regulated by RNA Polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we identified that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we find that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of Paf1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.
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Project description:Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is often upregulated in cancer to facilitate rapid cell growth and proliferation, and has emerged as a potential target for chemotherapeutic agents. BMH-21 and Pt(II) chemotherapeutic agent oxaliplatin are well documented as inhibitors of Pol I activity, however the underlying mechanisms for this inhibition are not completely understood. Here, we applied chromatin immunoprecipitation sequencing (ChIP-seq) techniques and immunofluorescence imaging to probe the influence of oxaliplatin and BMH-21 on Pol I machinery. We demonstrate oxaliplatin and BMH-21 induce early nucleolar stress leading to the formation of “nucleolar caps” containing Pol I and upstream binding factor (UBF) which corresponds with broad reductions in ribosomal DNA (rDNA) occupancy of Pol I. Distinct occupancy patterns for the two compounds are revealed in ChIP-seq experiments. Taken together, our findings suggest that in vivo, oxaliplatin does not induce Pol I inhibition via interrupting a specific step in Pol I transcription , while treatment with BMH-21 induced unique polymerase stalling at the promoter and terminator regions of the human ribosomal RNA gene.
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Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans.
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Project description:Chromatin places fundamental physical constraints on transcription (Gamarra and Narlikar, 2021). The PAF1 complex (PAF1C), a hexamer of PAF1, LEO1, CTR9, SKI8, CDC73 and RTF1, plays a critical role in transcription with incompletely understood mechanisms. During transcriptional elongation, PAF1C is one of the positive elongation factors in complex with RNA polymerase II (Pol II) to facilitate elongation through chromatin (Vos et al., 2018), but it is unclear yet if negative elongation factors are needed concurrently to restrain elongation. Here we show that besides decreasing elongation rate, LEO1 knockout in human K562 cells increases transcriptional readthrough and cellular level of C-terminal domain (CTD) phosphorylated Pol II while increases and decreases transcriptional output of several hundred genes, respectively. Mechanistic analyses taking proteomic, functional genomic and biochemical approaches discovered that PAF1C regulates transcriptional termination in part through recruiting PNUTS-PP1γ complex and facilitates Pol II transcriptional re-initiation through recruiting TOX4-PP1α complex. Moreover, Paf1 conditional knockout in mice severely blocks T cell development, increases cellular level of CTD phosphorylated Pol II, mainly decreases Pol II occupancy and transcriptional output in double positive T cells, and importantly, the regulation of re-initiation by PAF1C through TOX4-PP1α complex is conserved between mouse and human. Our results also suggest that PNUTS-PP1γ and TOX4-PP1α bind PAF1C to restrain Pol II elongation through chromatin. These findings not only establish PAF1C as a critical regulator of transcriptional termination and re-initiation besides elongation but also advanced current understanding of elongation.
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