Abstract
BackgroundThe co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR. However, the functional impact of this modification is not fully understood. Here, we investigated the influence of HuR methylation by CARM1 upon the turnover of HuR target mRNAs encoding senescence-regulatory proteins.ResultsChanging the methylation status of HuR in HeLa cells by either silencing CARM1 or mutating the major methylation site (R217K) greatly diminished the effect of HuR in regulating the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16. Although knockdown of CARM1 or HuR individually influenced the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16, joint knockdown of both CARM1 and HuR did not show further effect. Methylation by CARM1 enhanced the association of HuR with the 3′UTR of p16 mRNA, but not with the 3′UTR of cyclin A, cyclin B1, c-fos, or SIRT1 mRNAs. In senescent human diploid fibroblasts (HDFs), reduced CARM1 was accompanied by reduced HuR methylation. In addition, knockdown of CARM1 or mutation of the major methylation site of HuR in HDF markedly impaired the ability of HuR to regulate the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16 as well to maintain a proliferative phenotype.ConclusionCARM1 represses replicative senescence by methylating HuR and thereby enhancing HuR’s ability to regulate the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs.
Highlights
The co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR
By using two approaches to mimic the hypomethylation of HuR, knockdown of CARM1 and mutation of HuR at the major methylation site (R217), we have investigated the functional impact of the CARM1mediated HuR methylation and the mechanisms underlying in replicative senescence
Methylation by CARM1 enhances the effect of HuR in regulating the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs Because HuR has been reported to stabilize cyclin A, cyclin B1, c-fos, and SIRT1 mRNAs, and destabilize p16 mRNA [5,6,7], we asked if CARM1-mediated HuR methylation influences the expression of these genes
Summary
The co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR. We investigated the influence of HuR methylation by CARM1 upon the turnover of HuR target mRNAs encoding senescence-regulatory proteins. The molecular events controlling HuR function are not fully understood, the cytoplasmic presence and post-translational modification (e.g., phosphorylation and methylation) of HuR are important [2,9,10,11]. The phosphorylation of HuR at S221 by both PKCα and PKCδ is linked to HuR shuttling as well as the stabilization of COX-2 mRNA [12] These findings underscore functional links between HuR and the aforementioned signaling cascades, which regulate HuR shuttling or its binding to target mRNAs
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