Progress towards High-Throughput Approaches to Study Interactions of Demethylase Proteins and Epitranscriptomic RNA Modifications

Abstract

Post-transcriptional RNA modifications have been found to have a strong regulatory role through epitranscriptomics due to the interactions of “writers”, “readers” and “erasers” of these modifications. Of particular interest is the N6-methyladenosine (m6A) modification, which has been implicated in many critical biological processes. The m6A modification on RNA has been found to be enriched in the GG[m6A]CU motif, suggesting a possible sequence specificity of the enzymes that interact with this modification. Two known demethylases of this enzyme, ALKBH5 and FTO, have significant biological as well as clinical significance and understanding what guides their substrate specificity is essential for delineating the function. This study provides progress towards a larger goal of using a high-throughput chemical approach to probe the sequence specificity of m6A demethylases. By studying the activity of ALKBH5 and FTO towards a random m6A containing RNA library using an HPLC based demethylation assay, this study showed that ALKBH5 and FTO show selective activity, and only demethylate a small subset on the library, and ALKBH5 is more active than FTO. In addition, kinetic analysis of the demethylases, shows that ALKBH5 also shows faster rates of demethylation. Further, this analysis provides points to isolate the RNA libraries at different levels of demethylation, and thus characterize preferred sequences. Lastly, a protocol to isolate the substrates of demethylases from a random library of RNA sequences was developed and validated. These results together provide an approach to further the understanding of what guides the promiscuity and specificity of demethylase- m6A RNA modification interactions