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Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

ABSTRACT: Negative elongation factor (NELF) is a transcriptional regulator that is primarily known for its role in the promoter-proximal pausing of RNA polymerase II (RNAPII) [1-2]. We have previously demonstrated that acute depletion of the NELF subunit NELF-C causes genome-wide RNAPII pausing at a second site associated with the +1 nucleosome [3]. Here, we show that acute depletion of the NELF subunit NELF-C results in a reversible cellular senescence phenotype that includes impaired growth, robustly detectable β-galactosidase activity, and upregulated expression of senescence-associated genes. We also identify a small set of genes at which NELF-C depletion induces RNAPII release rather than second site pausing. We identify Elongin A (ELOA) as a transcription factor mediating the impacts of NELF depletion, and we demonstrate that the primate-specific ELOA homolog ELOA3 can also play this role. Mechanistically, ELOA mediates the impacts of NELF depletion at least in part by altering the kinetic coupling between the elongation and splicing of nascent transcripts, which results in intron retention and other splicing defects. Despite reports implicating splicing defects in aging-related phenomena [4-6] [7-9], mechanisms have remained elusive. The findings presented here indicate that the crosstalk between splicing factors, ELOA, and NELF may be critical to understanding cellular senescence and aging

ORGANISM(S): Homo sapiens

PROVIDER: GSE269718 | GEO | 2025/12/22

REPOSITORIES: GEO

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Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

Project description:Elongation factors control post-initiation steps of transcription by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, and SPT6 via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of elongation control and mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA KO suppresses NELF depletion-induced mRNA processing defects and the 3’ extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3’ end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic ELOA loss confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription termination-coupled processes, and links these to the complex phenomena of cellular senescence and aging.

2025-12-22 | GSE269715 | GEO

Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

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Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

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Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

Project description:This SuperSeries is composed of the SubSeries listed below. Elongation factors control post-initiation steps of transcription by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, and SPT6 via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of elongation control and mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA KO suppresses NELF depletion-induced mRNA processing defects and the 3’ extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3’ end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic ELOA loss confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription termination-coupled processes, and links these to the complex phenomena of cellular senescence and aging.

2025-12-22 | GSE269948 | GEO

Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes [RNA-seq]

Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Genome-wide expression in HeLa cells in the absence of Integrator 11, or NELF or mock (control) depleted by strand-specific RNASeq (Illumina)

2015-05-19 | E-GEOD-60585 | biostudies-arrayexpress

Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation

Project description:The human Negative Elongation Factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage in vitro. NELF-mediated stalling of RNAPII also attenuates transcription of a number of inducible genes in human cells. To obtain a genome-wide understanding of human NELF-mediated transcriptional regulation in vivo, we carried out an exon-array study in T47D breast cancer cells with transient siRNA knockdown of individual NELF subunits. Upon depletion of NELF-A, -C, or -E, the vast majority of NELF-regulated genes were down-regulated. Many of the down-regulated genes encode proteins that play key roles in cell cycle progression. Consequently, NELF knockdown resulted in significant reduction in DNA synthesis and cell proliferation. Chromatin immunoprecipitation showed that NELF knockdown led to dissociation of RNAPII from the promoter-proximal region of the cell cycle-regulating genes. This was accompanied by decreased histone modifications associated with active transcription initiation (H3K9Ac) and elongation (H3K36Me3), as well as reduced recruitment of the general transcription factor TFIIB and increased overall histone occupancy at a subset of the down-regulated promoters. Lastly, our study indicates that NELF regulates alternative transcription initiation of Basigin gene (BSG) by differentially influencing RNAPII density at the two neighboring exons at the 5’ end of the gene. Taken together, our data suggest a diverse transcriptional consequence of NELF-mediated RNAPII pausing in the human genome.

2010-01-20 | GSE19940 | GEO

Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes [ChIP-seq]

Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Binding profiles of Integrator subunits in HeLa cells by ChIP-Seq (Illumina) Please note that the MACS14*.tar.gz contains MACS output bed and xls files and the 'readme.txt' contains a detailed description of each file.

2015-05-19 | E-GEOD-60584 | biostudies-arrayexpress

TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription

Project description:Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNAPII may therefore have a fundamental impact in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show evidence for in vitro and in vivo interaction of TCERG1 with the Bcl-x pre-mRNA. Transcription profile analysis reveals that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the pro-apoptotic Bcl-xS 5’ splice site. ChIP-Seq

2012-01-12 | E-GEOD-33716 | biostudies-arrayexpress

Elongin A displays widespread association with actively transcribed genes and modulates enhancer RNA transcripiton.

Project description:Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes both to thousands of Pol II transcribed genes with a preference for sites of Pol II pausing and as well as a large number of active enhancers across the genome. EloA loss results in accumulation of transcripts at a subset of enhancers and their adjacent genes. However, EloA deletion results in only a modest decrease in the average elongation rate of Pol II globally. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interferes with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on eRNA synthesis and interacts with the rRNA producing/processing machinery in the nucleolus.

2020-12-10 | GSE151582 | GEO

SPT6 functions in transcriptional pause/release via PAF1C recruitment

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.

2022-08-09 | PXD033771 | Pride

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