The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [eCLIP]
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ABSTRACT: Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
ORGANISM(S): Human gammaherpesvirus 8 Homo sapiens
Project description:Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
Project description:Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
Project description:Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
Project description:In this study, we found that the host protein PNUTS suppressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene expression during lytic reactivation and from heterologous viral reporters. To gain insights into the mechanism, we performed ChIP-seq of the pol II subunit RPB3 in cells with an integrated reporter containing the KSHV ORF59 gene. Our results were consistent with previous reports showing that depletion of PNUTS leads to global loss of pol II slow down and termination after polyadenylation sites. In contrast, PNUTS depletion appeared to decrease promoter-proximal pausing on our integrated reporter suggesting a distinct mode of gene regulation on viral genes.
Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.
Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the mouse PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, these findings reveal an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Pol II-dependent transcription.
Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the mouse PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, these findings reveal an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Pol II-dependent transcription.
Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, this reveals an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Poll II-dependent transcription.
Project description:Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the Spt5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of non-coding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.
Project description:RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery. Conversely, the corresponding phosphatases, notably PP2A and PP1—the most abundant serine-threonine phosphatases in cells—are thought to mainly impede polymerase progression, respectively restraining pause release at promoters and polymerase elongation at terminators. Here we reveal an unexpected role of PP1, within the PNUTS-PP1 complex, in sustaining global transcriptional activation. Acute disruption of PNUTS-PP1 leads to severe defects in the release of paused polymerase and subsequent downregulation for the majority of transcribed genes. Mechanistically, PNUTS-PP1 promotes pause release by dephosphorylating multiple substrates, including the 7SK snRNP subunit MEPCE, a known regulator of pause release. PNUTS-PP1 exhibits antagonistic functions compared to INTAC phosphatase, which generally inhibits pause release. Our research thus highlights the opposing roles of PP1 and PP2A in modulating genome-wide transcriptional pausing and gene expression.