Characterization of novel methyltransferases METTL22 and FAM86A.1

Abstract

Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a 10 member family of sequences with high structural similarity consisting, indicating that these proteins may also have MTase activity. For 4 of the family members, recombinant constructs were made, expressed and batch purified before the focus was narrowed down to 2 for closer examination – FAM86A.1 and METTL22. GST-FAM86A.1 was purified to high purity and yield, and subsequently used for generation of antibodies. Analysis of the antisera showed that at least one of the antibodies is highly specific and useful in western blotting as well as in immunoprecipitation. In addition, conserved residues known to be involved in SAM binding were mutated in His-FAM86A.1 and His-METTL22, which completely abolished the methyltransferase activity demonstrated for the wild type proteins. Thermal shift assay confirmed the structural integrity of FAM86A.1 mutants, thus indicating that the loss of methyltransferase activity was due to lack of capacity to bind SAM.