Defining substrate specificities of human <scp>RNA</scp> capping methyltransferases through quantitative assessment of independent yet cooperative activities

Human RNA capping is critical for mRNA splicing, protection of RNA from 5' exonucleases in the cytoplasm, and targeting to the ribosome. Human RNMT, CMTR1, and CMTR2 are RNA methyltransferases involved in the RNA capping process. They play a significant role in the proliferation and differentiation of embryonic stem cells and have been implicated in cancer. Substrate specificities of human RNA capping methyltransferases have been somewhat explored in a few studies. Here, we report on a comprehensive, systematic, and quantitative assessment of their substrate specificities along with SARS-CoV-2 counterparts, nsp14 and nsp16. We discovered novel cooperative activities of human enzymes. We designed and synthesized various RNA substrates with defined patterns of methylation to systematically assess the dependency or cooperativity of their activities using radiometric assays followed by mass spectrometry to verify RNA methylation status. We have tested all five enzymes in parallel against these substrates and determined kinetic parameters. Our data not only indicate that the catalytic activities of human RNMT, CMTR1, and CMTR2 are distinct and nonoverlapping, but also provide a novel quantitative assessment of their activities, indicating how and to what extent these proteins affect each other's function. Unlike nsp14 and nsp16, their functions are not necessarily sequential, but show significant cooperativity. Altogether, our data provide a comprehensive understanding of substrate specificities of human RNA capping methyltransferases, enabling the development of potential future anticancer therapeutics and assessment of antiviral therapeutics' selectivity.