PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

Lisa-Marie Appel,Carmen Ebenwaldner,Rushad Pavri,Anna Vlasova,Richard Štefl ,Melania Bruno,Kristina Djinović-Carugo ,Irina Grishkovskaya,Gen Lin,Vedran Franke,Sebastian Kostrhon,Martin Puchinger ,Ursula E Schoeberl,Marek Šebesta ,Alexander Stark,Tanja Kaufmann,Altuna Akalin,Carrie Bernecky,Karl Mechtler,Etienne Beltzung,Martin Leeb,Aistė Kasiliauskaitė ,Dea Slade

doi:10.1038/s41467-021-26360-2

Abstract

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

Highlights

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing
Neither the TFIIS-Like Domain (TLD) nor Plant Homeo Domain (PHD) domains were required for the interaction between Pol II and FLAG-PHF3 (Fig. 1f), which was unexpected given that the TLD is required for the Bypass of Ess-1 (Bye1)-Pol II interaction in yeast[31,34]
We found that PHF3 binds to the Pol II CTD phosphorylated on S2 through a CTD-binding domain called Spen Paralogue and Orthologue C-terminal (SPOC)

Summary

Introduction

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Transcription is tightly coupled with cotranscriptional RNA processing whereby Pol II CTD acts as a docking site for 5’ mRNA capping, splicing, 3’end processing, termination and mRNA export factors that recognize specific CTD phosphorylation patterns[10,21,22,23]. MRNA capping enzymes such as Cgt[1], Pce[1] and Mce[1] were shown to employ the nucleotidyltransferase (NT) domain to directly bind pS5 within the Pol II CTD, whereas 3’end processing and termination factors such as yeast Pcf[11] and mammalian

Methods

Results

Conclusion