Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation.

Maxim I Maron,Varun Gupta,Alyssa D Casill,Jacob S Roth,Charles C Query,Matthew J Gamble,David Shechter,Simone Sidoli

doi:10.7554/elife.72867

Abstract

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

Highlights

The mammalian genome encodes nine protein arginine methyltransferases (PRMTs 1-9; PRMT4 is known as CARM1)
Type I Protein arginine methyltransferases (PRMTs) further catalyze the formation of asym98 metric NG,NG-dimethylarginine (Rme2a); Type II PRMTs (PRMT5 and 9) form symmetric 99 NG,N’G-dimethylarginine (Rme2s)
As previous reports indicated that lengthy treatment with PRMT inhibitors promotes aberrant RNA splicing, we wanted to determine whether alternative splicing differences occurred as early as day two and, if so, how they changed over time (Bezzi et al 2013; Fong et al 103 2019; Radzisheuskaya et al 2019; Tan et al 2019; Li et al 2021; Sachamitr et al 2021)

Summary

Introduction

The mammalian genome encodes nine protein arginine methyltransferases (PRMTs 1-9; PRMT4 is known as CARM1). Core components of the spliceosome (Meister et al 2001; Neuenkirchen et al 2015) This includes both non-enzymatic chaperoning of Sm proteins via PRMT5/pICln following their translation and post-translational methylation of SmD1 (SNRPD1), SmD3 (SNRPD3), and SmB/B’. (SNRPB), by PRMT5-MEP50 (Friesen et al 2001; Meister et al 2001; Paknia et al 2016) Following their methylation, these Sm proteins are delivered to the Survival of Motor Neuron (SMN) assembly factor, by which they are bound to small nuclear RNAs (snRNAs) in preparation for further processing and eventual nuclear import (Boisvert et al 2002; Meister and Fischer 2002; Pellizzoni et al 2002). Determining how PRMT activity governs intron retention is of great interest

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