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U1 snRNP complex with cleavage and polyadenylation factors controls mRNA transcription termination

ABSTRACT: Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3’-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1–CPAFs), that binds intronic PASs and suppresses PCPA. U1–CPAFs are distinct from U1-spliceosomal complexes; they include CPA’s three main subunits, CFIm, CPSF, and CstF, lack essential splicing factors, and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1–CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1–CPAFs’ switch from repressive to activated states. Our findings outline U1 telescripting mechanism and demonstrate U1’s unique role as central-regulator of pre-mRNA processing and transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE135140 | GEO | 2019/07/31

REPOSITORIES: GEO

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Project description:Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle or transcription, and are emerging as promising therapeutic targets in cancer cells. The transcriptional kinase, CDK12, is especially intriguing, as it modulates transcription elongation by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase (Pol II) with subsequent effects on expression of DNA damage response (DDR) genes. Yet the exact mechanism by which CDK12 regulates this vital process remains unclear. Using the selective inhibitor of CDK12/13, THZ5317 and transient transcriptome sequencing (TT-seq), we sought to capture the dynamic transcriptional changes linked to CDK12 by analyzing the immediate, early effects on nascent RNA production after CDK12 depletion in neuroblastoma (NB) cells. CDK12-induced inhibition resulted in gene length-dependent defects in transcription elongation, leading to a disproportionate loss of 3' reads in long genes. Short transcripts, by contrast, including those of replication-dependent histone genes, underwent 3' UTR extension. The effect on long genes was accompanied by premature cleavage and polyadenylation (PCPA), followed by early termination and loss of gene expression. DDR gene transcripts were the most prominent among those terminated by PCPA. Phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in CPA , are direct phospho-targets of CDK12 and that CDK depletion phenocopied their effects on transcription. Importantly, sensitivity to CDK12 inhibition was predicted by a lower proportion of U1 snRNP binding sites compared to polyadenylation sites (PAS) and hence a higher propensity to intronic poly(A) site selection - a characteristic observed in most DDR genes. These results support a model in which CDK12 regulates expression of DDR genes not only by regulating transcription elongation through its effects on Pol II CTD phosphorylation, but also through direct phosphorylation of pre-mRNA processing factors. These mechanistic insights may enable the development of new anti-cancer strategies targeting multiple vulnerable steps in CDK12-dependent regulation of DNA damage repair.

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U1 snRNP complex with cleavage and polyadenylation factors controls mRNA transcription termination

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