Characterization of the human putative methyltransferase SETD9

Protein methylation plays an important role in many cellular possesses, including gene regulation, where it targets histone proteins present in the nucleus. Histone methylation is one of the best-studied post-translational modifications. In recent years, several methyltransferases (MTases) that target non-histone proteins have also been characterized. These MTases have been found to impact several functional aspects of its target proteins, such as protein-protein interactions, and/or their enzymatic activities. Some of these MTases were found to exert their function in various organelles, such as the endoplasmic reticulum, mitochondria or ribosomes. There are still many putative MTases that need to be characterized, including several members of the superfamily of SET-domain containing MTases. Until recently, the SET proteins were generally considered to be only lysine-specific, i.e. to methylate lysine residues, and only to target histones. However, new research has characterized non-histone SET proteins such as SETD7, which is a promiscuous MTase that targets many proteins, or SETD3, which is histidine-specific and targets actin. According to bioinformatic analyzes, there are still a few non-histone methyltransferases that remain uncharacterized. Among them is SETD9, which has been predicted to reside in the mitochondria and exert its activity there. The main focus of this study was to determine the intracellular localization of SETD9 and find its biochemical function. Bioinformatics analysis identified that SETD9 has a broad albeit scattered evolutionary distribution, being present in most vertebrates, but also in selected invertebrate animals. Sequence comparison of SETD9 to other human SET proteins revealed the presence of several, but not all, sequence motifs typical for SET MTases, which are required for their enzymatic activity and binding of S-adenosylmethionine cofactor. Using fluorescence microscopy of GFP-tagged SETD9, it was confirmed that human SETD9 is a mitochondrial protein and that its most N-terminal region is an autonomous mitochondrial targeting sequence, which seems to be mainly retained in the mature protein. Expression of TAP-tagged SETD9 inside cells made it possible to pull down proteins that co-purified with SETD9, these may represent intracellular binding partners. Moreover, a radioactivity-based enzymatic MTase assay was used to determine whether SETD9 was an active MTase, able to methylate proteins present in cellular extracts. However, the results of these experiments were inconclusive. Thus, further research is needed to determine if SETD9 truly represents an active MTase, and if so, identify its biological targets.