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The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription

ABSTRACT: PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

SUBMITTER: Dr. Ahmet C. Berkyurek

PROVIDER: S-SCDT-EMBOJ-2020-105565 | biostudies-other |

REPOSITORIES: biostudies-other

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The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

Project description:PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

| S-EPMC7917558 | biostudies-literature

Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors.

Project description:Piwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from > 15,000 discrete genomic loci by RNA polymerase II (Pol II), resulting in 28 nt short-capped piRNA precursors. Here, we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. Moreover, we demonstrate that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Taken together, our results draw new parallels between snRNA and piRNA biogenesis in nematodes and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II during piRNA biogenesis.

| S-EPMC7917550 | biostudies-literature

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

Project description:The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

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The RNA Polymerase II subunit RPB-9 directs transcriptional elongation at piRNA loci in C. elegans

Project description:PIWI-interacting RNAs (piRNAs) are genomically-encoded small RNAs that regulate germ cell development and guarantee germline integrity. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. To date, piRNA biogenesis mechanisms are still unclear. Here, we show that the RNA Polymerase II subunit RPB-9 is required to promote transcription elongation at piRNA loci. Through genetic and biochemical experiments, we demonstrate that rpb-9-mediated piRNA production is needed to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline.

2020-12-07 | PXD018657 | Pride

Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes.

Project description:RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 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 INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

| S-EPMC4263189 | biostudies-literature

Histone H3R17me2a mark recruits human RNA polymerase-associated factor 1 complex to activate transcription.

Project description:The histone coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator for a number of transcription factors, including nuclear receptors. Although CARM1 and its asymmetrically deposited dimethylation at histone H3 arginine 17 (H3R17me2a) are associated with transcription activation, the mechanism by which CARM1 activates transcription remains unclear. Using an unbiased biochemical approach, we discovered that the transcription elongation-associated PAF1 complex (PAF1c) directly interacts with H3R17me2a. PAF1c binds to histone H3 tails harboring dimethylation at R17 in CARM1-methylated histone octamers. Knockdown of either PAF1c subunits or CARM1 affected transcription of CARM1-regulated, estrogen-responsive genes. Furthermore, either CARM1 knockdown or CARM1 enzyme-deficient mutant knockin resulted in decreased H3R17me2a accompanied by the reduction of PAF1c occupancy at the proximal promoter estrogen-responsive elements. In contrast, PAF1c knockdown elicited no effects on H3R17me2a but reduced the H3K4me3 level at estrogen-responsive elements. These observations suggest that, apart from PAF1c's established roles in directing histone modifications, PAF1c acts as an arginine methyl histone effector that is recruited to promoters and activates a subset of genes, including targets of estrogen signaling.

| S-EPMC3326481 | biostudies-literature

The RNA Polymerase II subunit RPB-9 directs transcriptional elongation at piRNA loci in C. elegans

2020-12-04 | GSE149071 | GEO

Architecture of an RNA polymerase II transcription pre-initiation complex.

Project description:The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.

| S-EPMC4039082 | biostudies-literature

Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.

Project description:Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.

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Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex.

Project description:The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit. The Paf1-Leo1 heterodimer and Cdc73 form opposite ends of Paf1C, whereas Ctr9 bridges between them. Consistent with the structural observations, the initiation factor TFIIF impairs Paf1C binding to Pol II, whereas the elongation factor TFIIS enhances it. We further show that Paf1C is globally required for normal mRNA transcription in yeast. These results provide a three-dimensional framework for further analysis of Paf1C function in transcription through chromatin.

| S-EPMC5467213 | biostudies-literature

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