Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing

Abstract

Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator FLOWERING LOCUS KH DOMAIN (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor FLOWERING LOCUS C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper premRNA splicing that impacts diverse developmental processes. P RMT5 is a type II protein arginine methyltransferase that catalyzes the formation of monomethylarginine and symmetric ω-N G , N′ G -dimethylarginine (SDMA) (1). PRMT5 is highly conserved among yeast, animals, and higher plants, and is implicated in diverse cellular and biological processes, including transcriptional regulation, RNA metabolism (1, 2), ribosome biogenesis (3), Golgi apparatus structure maintenance (4), and cell cycle regulation (5). In mouse, fly, and worm, PRMT5 orthologs are involved in germ-cell formation, specification, and maintenance (6-10).In mammalian cells, PRMT5 localizes to both the cytoplasm and the nucleus, and methylates multiple histone and nonhistone proteins (1). In the nucleus, PRMT5 usually functions in repressing target genes by methylating histone H4 Arginine 3 (H4R3), H3R8, as well as transcription factors/regulators (5, 11, 12). In the cytoplasm, PRMT5 mainly exists in a 20S methylosome complex, consisting of spliceosomal U snRNP (uridinerich small nuclear riboucleoprotein particles) Sm proteins (including Sm B/B′, D1, D2, D3, E, F, and G), PRMT5, pICln, and WD repeat protein/MEP50 (13-15). In this complex, U1, 2, 4, 5 snRNP Sm proteins Sm B/B′, D1, D3, and U6 snRNP-specific Sm-like protein LSm4 are symmetrically dimethylated by PRMT5 (13,16). Such methylation can increase the binding affinity of these Sm proteins for the downstream recipient, Survival Motor Neuron, the spinal muscular atrophy disease gene product (17,18). Subsequently, the PRMT5-and Survival Motor Neuroncom...

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