Coactivator-associated Arginine Methyltransferase 1, CARM1, Affects Pre-mRNA Splicing in an Isoform-specific Manner*♦

Abstract

Molecular diversity through alternative splicing is important for cellular function and development. However, little is known about the factors that regulate alternative splicing. Here we demonstrate that one isoform of coactivator-associated arginine methyltransferase 1 (named CARM1-v3) associates with the U1 small nuclear RNP-specific protein U1C and affects 5 splice site selection of the pre-mRNA splicing. CARM1-v3 was generated by the retention of introns 15 and 16 of the primary transcript of CARM1. Its deduced protein lacks the C-terminal domain of the major isoform of CARM1 and instead has v3-specific sequences at the C terminus. CARM1-v3, but not the other isoforms, strongly stimulates a shift to the distal 5 splice site of the pre-mRNA when the adenoviral E1A minigene is used as a reporter and enhances the exon skips in the CD44 reporter. A CARM1-v3 mutant lacking the v3-specific sequences completely lost the ability to regulate the alternative splicing patterns. In addition, CARM1-v3 shows tissuespecific expression patterns distinct from those of the other isoforms. These results suggest that the transcriptional coactivator can affect the splice site decision in an isoform-specific manner.It has been estimated that about 60% of human genes undergo alternative splicing (1). Commonly, alternative splicing determines the inclusion of a portion of coding sequence in the mRNA, giving rise to protein isoforms that differ in their peptide sequence and hence chemical and biological activity. The mechanism of alternative splicing permits diversity of translatable mRNAs, thereby increasing the proteome diversity encoded by a limited number of genes. Genetic switches based on alternative splicing are known to be important in many cellular and developmental processes, including sex determination, apoptosis, axon guidance, and tissue-specific differentiation (2-4).Although a large variety of splicing decisions can be explained by the antagonistic effects of general splicing factors, such as serine/arginine-rich proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), 1 it is most likely that tissue-specific or developmentally regulated splicing factors have an important role in the regulation of alternative splicing. In Drosophila melanogaster, sex-lethal functions as a regulator of alternative splicing in sex determination (2), and the embryonic lethal abnormal visual system is a gene-specific regulator of alternative pre-mRNA processing in neurons (3). However, in mammals, only a limited number of splicing regulators have been identified to date, despite the large diversity of the mammalian gene transcripts, and little is known about the mechanisms by which alternative splicing is regulated.Pre-mRNA splicing occurs in a large multicomponent ribonucleoprotein complex called the spliceosome, which is composed of U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein (snRNP) particles and many non-snRNP protein splicing factors (5). U1 snRNP recognizes the 5Ј splice site and is among the first factors to inter...

Alternative Splicing Yields Protein Arginine Methyltransferase 1 Isoforms with Distinct Activity, Substrate Specificity, and Subcellular Localization

“…In addition to PRMT1, alternative splicing was also reported for CARM1 and PRMT7 pre-mRNAs (77,78), although extensive biochemical characterization of the respective PRMT isoforms generated is still lacking. As we have shown in the current study, alternative splicing can lead to the production of PRMT variants with a wide array of different biochemical and regulatory properties.…”

Histone H3R17me2a mark recruits human RNA Polymerase-Associated Factor 1 Complex to activate transcription

“…PAF1c functions in transcription initiation, elongation (15), mRNA 3′ end cleavage (25), polyadenylation of mRNA precursors (27), and small nucleolar RNA (snoRNA) 3′ end formation (31). In addition to transcriptional activation, CARM1 methylates splicing factors (32), modulates recognition of 5′ splicing sites (33), and regulates the coupling of transcription and splicing (34). Furthermore, a recent report showed that RNAPII carboxyl-terminal domain is methylated by CARM1 (35).…”

CARM1 Regulates Proliferation of PC12 Cells by Methylating HuD

“…The underlined sequences form a stem region of CARM1 shRNA which corresponds to 229 to 247 nucleotides of rat CARM1. This sequence is shared by all of the reported splicing variants of rat CARM1 (51). To generate stable cell lines, neomycin-resistant gene cassettes should be added to pSILENCER-shCARM1 by recloning BamHI-XbaI digested fragments containing the U6 promoter and CARM1 shRNA sequence into pcDNA3.1 vector with BglII and XbaI sites (pSILENCER-shCARM1 Neo r ).…”