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WDR82 Code Coordinates Transcription and pre-mRNA 3’-End Processing Through Regulated Phosphorylation

ABSTRACT: Phosphorylation plays a central role in coordinating transcription with pre-mRNA maturation, yet the mechanistic understanding of how transcription is regulated by phosphorylation remains limited. The PP1 phosphatase removes CDK9-dependent phosphorylation of Pol II and Spt5, triggering transcription termination. Here, we demonstrate that PNUTS enhances PP1 activity, whereas WDR82 mediates an interaction between PP1 and the pre-mRNA 3’-end processing machinery subunit Symplekin. We present the first structure of a PNUTS-WDR82-Symplekin-Ssu72 complex, revealing that PNUTS and Symplekin bind WDR82 through distinct interfaces using conserved short linear motifs. Mutations that inactivate PP1's catalytic activity or disrupt its interaction with either PNUTS or the 3’-end processing machinery led to impaired transcription elongation, inefficient cleavage of nascent transcripts, altered poly(A) site selection, and widespread transcription termination defects. Our findings demonstrate that the PP1 complex is essential for coupling transcription to pre-mRNA 3’-end maturation, ensuring production of functional mRNA.

INSTRUMENT(S):

ORGANISM(S): Schizosaccharomyces Pombe

SUBMITTER: Marjorie Fournier

LAB HEAD: Lidia Vasilieva

PROVIDER: PXD081886 | Pride | 2026-07-30

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	PNUTS-FTP-TANDEM.csv	Csv
	PPW-Symplekin-Crosslinking.csv	Csv
	PPW_Stablecomplex.csv	Csv
	QEX02_MF_210224_HA-XL_159.raw	Raw
	QEX03_AA_220111_Ppn1_068.raw	Raw

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PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA pol II transcription termination

Project description:The restrictor, ZC3H4/WDR82, terminates antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 immunoprecipitate with PP1 phosphatase and its nuclear targeting subunit, PNUTS, which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS with restrictor where both PNUTS and ZC3H4 contact WDR82. A substrate trap, PP1H66K-PNUTS, comprising inactive PP1 fused to the PNUTS C-terminus antagonizes restrictor mediated termination whereas PP1WT-PNUTS has less effect suggesting that phosphatase activity is required for termination. One PP1/PNUTS substrate implicated in termination by restrictor is pol II CTD Ser5-P. PP1H66K-PNUTS induces Ser5-P hyperphosphorylation at 5’ ends presumably by inhibiting dephosphorylation. NET-seq analysis suggests that CTD Ser5 dephosphorylation would promote termination by increasing pol II pausing. Both inhibition of termination and CTD hyperphosphorylation require the WDR82 binding domain of PP1H66K-PNUTS that mediates restrictor binding. In summary, the PP1/PNUTS phosphatase associated with restrictor via WDR82 promotes efficient transcription termination.

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Project description:The restrictor complex, WDR82/ZC3H4, is the major termination factor for antisense transcription from bidirectional promoters, but its mechanism of action is poorly understood. We report that the PP1 phosphatase nuclear targeting subunit PNUTS binds to restrictor via its WDR82 subunit. AlphaFold predicts a quaternary complex, PPWZ, in which PP1-associated PNUTS and ZC3H4 both contact WDR82. A chimera comprising inactive PP1H66K fused to C-terminal PNUTS sequences is a dominant-negative inhibitor of antisense termination and CTD Ser5 dephosphorylation and both activities require the PNUTS WDR82 binding domain. PP1H66K-PNUTS is a substrate trap that also interacts with pol II large subunit and nuclear exosome components. Ser5 hyperphosphorylated pol II has increased processivity and pauses less frequently than total pol II. We propose that the elevated elongation efficiency of Ser5-P pol II antagonizes early termination and that Ser5 dephosphorylation by PP1/PNUTS/WDR82/ZC3H4 is coupled to termination of antisense transcription.

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