Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

Abstract

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is involved in a multitude of biological processes in eukaryotes. Symmetric arginine dimethylation mediated by PRMT5 modulates constitutive and alternative pre-mRNA splicing of diverse genes to regulate normal growth and development in multiple species; however, the underlying molecular mechanism remains largely unknown. A genetic screen for suppressors of an Arabidopsis symmetric arginine dimethyltransferase mutant, atprmt5, identified two gain-of-function alleles of pre-mRNA processing factor 8 gene (prp8-8 and prp8-9), the highly conserved core component of the U5 small nuclear ribonucleoprotein (snRNP) and the spliceosome. These two atprmt5 prp8 double mutants showed suppression of the developmental and splicing alterations of atprmt5 mutants. In atprmt5 mutants, the NineTeen complex failed to be assembled into the U5 snRNP to form an activated spliceosome; this phenotype was restored in the atprmt5 prp8-8 double mutants. We also found that loss of symmetric arginine dimethylation of Sm proteins prevents recruitment of the NineTeen complex and initiation of spliceosome activation. Together, our findings demonstrate that symmetric arginine dimethylation has important functions in spliceosome assembly and activation, and uncover a key molecular mechanism for arginine methylation in pre-mRNA splicing that impacts diverse developmental processes.arginine methylation | protein arginine methyltransferase | AtPRMT5 | pre-mRNA splicing | Prp19C/NTC P rotein arginine methyltransferase 5 (PRMT5), a highly conserved type II protein arginine methyltransferase, transfers methyl groups to arginine residues, generating monomethylarginine and symmetric ω-N G , N′ G -dimethylarginine (SDMA) (1-3). In mammals, PRMT5 methylates and interacts with diverse proteins, including histones and other proteins, and has long been implicated in the modulation of a variety of processes, such as transcriptional regulation, RNA metabolism (4), apoptosis (5), signal transduction (6), and germ cell development (7). Both loss-of-function and overexpression of PRMT5 are fatal in mammals (8). AtPRMT5, the Arabidopsis homolog of PRMT5, regulates multiple aspects of plant growth and development, such as flowering time, growth rate, leaf morphology, sensitivity to stress conditions, and circadian rhythm, by modulating transcription, constitutive and alternative precursor mRNA (pre-mRNA) splicing of diverse genes (9-13). AtPRMT5 also mediates the symmetric arginine dimethylation of uridine-rich small nuclear ribonucleoproteins (U snRNPs) AtSmD1, D3, and AtLSm4 proteins, thus linking arginine methylation and splicing (10,11,13). However, the exact mechanism remains elusive.Pre-mRNA splicing occurs in the nucleus, removing introns and ligating exons (14). In eukaryotes, most introns are spliced in a series of reactions catalyzed by the spliceosome, which consists of five subcomplexes of U snRNPs and several non-snRNP factors. Each U snRNP contains distinct splicing...

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