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.
Highlights
Co-activator-associated arginine (R) methyltransferase 1 (CARM1), known as PRMT4, belongs to the type I protein arginine methyltransferase (PRMT) family that asymmetrically dimethylates protein substrates on arginines [1]
No deletion was found by sequencing genomic DNA, nor was any mutation identified in CARM1 mRNA derived from MCF7 cancer cells, implying that CARM1 in cancer cells is regulated by alternative splicing, resulting in exon 15 exclusion
We found that endogenous CARM1 protein isoforms (Figure 2E) largely mirrors the mRNA isoforms (Figure 2F) where CARM1 full-length (CARM1FL) is predominant in mouse brain, heart and muscle tissues, and both CARM1 isoform proteins could be detected in mouse lung, spleen and ovary tissues (Figure 2E)
Summary
Co-activator-associated arginine (R) methyltransferase 1 (CARM1), known as PRMT4, belongs to the type I protein arginine methyltransferase (PRMT) family that asymmetrically dimethylates protein substrates on arginines [1]. CARM1 methylates histone H3 at R2, R17 and R26 [4], which correlates with activation of ER-target gene pS2 [5]. Using a gain-of-function approach in ERa-positive breast cancer cells, we showed that 2-fold CARM1 overexpression in MCF7 cells led to growth inhibition, activation of differentiation markers and inhibition of anchorage-independent growth [8]. ERa regulates a number of genes that are essential for the etiology and progression of breast cancer. These findings suggest that CARM1 uniquely regulates growth inhibition and differentiation in ERapositive breast cancer cells through global regulation of ERa-regulated genes
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