The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.
Keywords: Cfr; Methyl adenosine; RNA methylation; Radical SAM; RlmN; Substrate identification; TGIRT.