Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Panmethylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA,-ADMA and-SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.

Automated summary

Arginine methylation is an important posttranslational modification, playing a crucial role in biological regulation and disease development. In this study, pan-methylarginine antibodies were generated using a novel antigen approach, enabling the observation of substrate scavenging by PRMT1 and the increased ADMA levels of splicing factor SmD1 upon PRMT5 inhibition. Additionally, blocking the catalysis of both SDMA and ADMA resulted in the loss of interaction between SmD1 and SMN. These findings contribute to a better understanding of the biological functions and regulatory mechanisms of arginine methylation.