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Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation.

ABSTRACT: Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation.

PROVIDER: PRJNA1231566 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28380165_1.fastq.gz	Fastqsanger.gz
	SRR28380165_2.fastq.gz	Fastqsanger.gz
	SRR28380166_1.fastq.gz	Fastqsanger.gz
	SRR28380166_2.fastq.gz	Fastqsanger.gz
	SRR28380167_1.fastq.gz	Fastqsanger.gz

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Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation [RNA-Seq]

Project description:The Piwi-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism behind this RNA-mediated epigenetic regulation that leads to heterochromatin formation is not yet fully understood. Through RNA interference screens, we discovered Pcf11 and PNUTS, two conserved termination factors of RNA polymerase II (Pol II), as essential for piRNA-guided silencing. When Pcf11 is artificially tethered to a reporter, it leads to co-transcriptional repression and Pol II stalling, both of which are dependent on an alpha-helical region of Pcf11 capable of forming condensates when triggered by the C-terminal repeat domain (CTD) of Pol II. Interestingly, an intrinsically disordered region (IDR) derived from the Plant FCA can substitute for the alpha-helical region of Pcf11 in its silencing capacity and support animal development, demonstrating a causal relationship between phase separation and Pcf11’s function. Further exploration into how Pcf11 causes Pol II stalling revealed a direct interaction between Pcf11’s CTD-interaction domain (CID) and a CTR region of Spt5. The dephosphorylation of Spt5-CTR by PP1/PNUTS is essential for Pcf11’s recruitment during Pol II termination. It appears that the phosphorylated state of CTR inhibits Pcf11 condensate formation, while the phosphorylation of Pol II CTD at Threonine 4 enhances Pcf11’s ability to undergo phase separation. In conclusion, we propose a model in which Pcf11 regulates transcriptional termination by slowing down Pol II elongation, a process that is aided by phase separation with the unphosphorylated Spt5. The ability of Pcf11 to stall Pol II, utilized by the piRNA pathway to initiate heterochromatin formation, might represent a universal strategy for nascent RNA-mediated epigenetic control.

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