Abstract
Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1RK. hnRNPUL1RK was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1RK had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1RK was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.
Highlights
Arginine methylation is a common post-translational modification that takes place in eukaryotic cells[1,2,3,4]
GFP-hnRNPUL1 was effectively recruited at sites of DNA damage, while GFP-hnRNPUL1RK was not recruited at these DNA damage sites with DRB treatment (Fig. 4). These findings suggest that arginine methylation of the RGG/RG motifs is required for the recruitment at sites of DNA damage in the presence of transcription inhibitors, but are not required for the exclusion of hnRNPUL1 from sites of DNA damage
In vivo and in vitro experiments confirmed that protein arginine methyltransferase 1 (PRMT1) was the enzyme responsible for the methylation of these RGG/RG sequences
Summary
Arginine methylation is a common post-translational modification that takes place in eukaryotic cells[1,2,3,4]. It is known that several DNA repair proteins including 53BP111,12, p5313, FEN114, BRCA115, RAD916, and DNA polymerase β are regulated by arginine methylation[17]. There are 3 main forms of methylated arginine identified in eukaryotes: ω -NG-monomethylarginine (MMA), ω -NG,NG-asymmetric dimethylarginine (aDMA), and ω -NG,N’G-symmetric dimethylarginine (sDMA). PRMT1 is the main type I mammalian enzyme with a preference for RGG/RG motifs[4] and embryos from PRMT1 knockout mice die shortly after implantation at E6.520,21. Non-coding RNAs, RNA helicases and RNA binding proteins (RBP) have recently been shown to participate in DNA damage signaling. RBPs including hnRNPK26, p54nrb/NONO27, hnRNPUL128, RBMX and DDX1729 have been identified as participants in the DDR pathway. The role and regulation of RNA, RBPs and ribonucleoprotein complexes at DSBs is unknown
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