Automethylation of CARM1 allows coupling of transcription and mRNA splicing

McArdle Laboratory for Cancer Research and Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706, USA.
Nucleic Acids Research (Impact Factor: 9.11). 12/2010; 39(7):2717-26. DOI: 10.1093/nar/gkq1246
Source: PubMed


Coactivator-associated arginine methyltransferase 1 (CARM1), the histone arginine methyltransferase and coactivator for many
transcription factors, is subject to multiple post-translational modifications (PTMs). To unbiasedly investigate novel CARM1
PTMs we employed high-resolution top-down mass spectrometry. Surprisingly, mouse CARM1 expressed in insect and mammalian expression
systems was completely dimethylated at a single site in the C-terminal domain (CTD). We demonstrate that dimethylation of
CARM1 occurs both in vivo and in vitro and proceeds via an automethylation mechanism. To probe function of automethylation, we mutated arginine 551 to lysine to
create an automethylation-deficient CARM1. Although mutation of CARM1's automethylation site did not affect its enzymatic
activity, it did impair both CARM1-activated transcription and pre-mRNA splicing. These results strongly imply that automethylation
of CARM1 provides a direct link to couple transcription and pre-mRNA splicing in a manner differing from the other steroid
receptor coactivators. Furthermore, our study identifies a self-regulatory signaling mechanism from CARM1's catalytic domain
to its CTD.

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    • "PRMT6 requires homodimerization to transfer a methyl group from SAM to the protein substrate and this could favour automethylation [27]. As documented for CARM1 (PRMT4), another member of the PRMT family, we have hypothesized that automethylation could modulate PRMT6 function [28]. Mutagenesis of the automethylation site of CARMI did not affect its catalytic activity but impaired its transcriptional and RNA-processing capacity. "
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    ABSTRACT: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown. Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays. These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.
    Full-text · Article · Jul 2013 · Retrovirology
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    • "Recently, a third phosphorylation site was identified at S448, which mediates the direct interaction of CARM1 with unliganded ERα to mediate ligand-independent activation of ERα (16). Finally, using top-down mass spectrometry, we mapped a single CARM1 automethylation site to R551 (in exon 15) in recombinant mouse protein, which is conserved among all vertebrate CARM1 proteins (17). Mutation of the automethylation site from arginine to lysine does not alter the enzymatic activity of CARM1. "
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    ABSTRACT: Co-activator-associated arginine methyltransferase 1 (CARM1) is subjected to multiple post-translational modifications. Our previous finding that automethylation of CARM1 is essential for regulation of transcription and pre-mRNA splicing prompted us to investigate how automethylation is regulated. Here, we report that automethylation is regulated by alternative splicing of CARM1 mRNA to remove exon 15, containing the automethylation site. Specifically, we find that two major alternative transcripts encoding full-length CARM1 (CARM1FL) and CARM1 with exon 15 deleted (CARM1ΔE15) exist in cells, and each transcript produces the expected protein. Further biochemical characterizations of the automethylation-defective mutant and CARM1ΔE15 reveal overlapping yet different properties. Interestingly, other arginine methylation substrates also have missing exons encompassing the site(s) of methylation, suggesting that protein arginine methylation level may, in general, be controlled by the alternative splicing mechanism. Finally, we observed differential distribution of CARM1FL and CARM1ΔE15 in epithelial and stromal cells in normal mouse mammary gland. Thus, alternative splicing not only serves as the determinant for CARM1 automethylation but also generates cell type-specific isoforms that might regulate normal ERα biology in the mammary gland.
    Full-text · Article · May 2013 · Nucleic Acids Research
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    • "Proteins involved in pre-mRNA splicing are also modified by methylation. The coactivator-associated arginine methyltransferase 1 (CARM1), which methylates H3 at R17 and is a transcriptional coactivator, methylates RNA binding proteins ELAV1/HuR, SNRPB/SmB, SNRPC/U1C and SF3B4/SAP49 (Kuhn et al., 2011). CARM1 has a role in alternative splicing. "
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    ABSTRACT: Epigenetics refer to a variety of processes that have long-term effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are histone modifications and DNA methylation which, in concert with chromatin remodeling complexes, nuclear architecture and microRNAs, define the chromatin structure of a gene and its transcriptional activity. There is a growing awareness that histone modifications and chromatin organization influence pre-mRNA splicing. Further there is emerging evidence that pre-mRNA splicing itself influences chromatin organization. In the mammalian genome around 95% of multi-exon genes generate alternatively spliced transcripts, the products of which create proteins with different functions. It is now established that several human diseases are a direct consequence of aberrant splicing events. In this review we present the interplay between epigenetic mechanisms and splicing regulation, as well as discuss recent studies on the role of histone deacetylases in splicing activities.
    Full-text · Article · Sep 2012
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