Biochemical Investigation into Novel Activities of Yeast SPOUT‐Superfamily RNA Methyltransferases

Abstract

The SPOUT superfamily of S‐adenosyl‐L‐methionine (SAM) dependent methyltransferases are involved in post‐transcriptional RNA modifications. Nep1, a SPOUT superfamily member, methylates rRNA at the N1 position of a post‐transcriptionally incorporated pseudouridine in the 18S rRNA of Saccharomyces cerevisiae (yeast) and in humans is implicated in cases of Diamond‐Blackfan anemia. We found that Nep1 can bind and methylate a specific pseudouridine found in the U2 snRNA in vitro, suggesting that Nep1 may also play a role in splicing regulation. Given this finding, other SPOUT proteins may also be able to modify snRNAs and be involved in splicing regulation. YGR283C and YMR310C are two other yeast SPOUT methyl transferases whose specific biological functions are not known but are known to interact with the U2 snRNP yeast protein, Lea1, and the yeast homolog of dyskerin, a protein involved in pseudouridylation of the U2 snRNA. We have expressed yeast YGR283C in E. coli and purified the protein for biochemical and structural characterization. Fluorescence polarization assays and electrophoretic mobility shift assays will be conducted to quantify YGR283C binding affinities for snRNAs. Filter binding assays will be used to quantify [3H]‐methyl incorporation from SAM into snRNA substrates. Together these studies will elucidate the function of these previously uncharacterized SPOUT superfamily members.