Lysine-specific methyltransferase Set7/9 (KMT7) belongs to the SET domain family of proteins. Besides the SET domain, Set7/9 also contains a so-called MORN (Membrane Occupation and Recognition Nexus) domain whose function in high eukaryotes is largely unknown. Set7/9 has been shown to specifically methylate both histones H1 and H3 as well as a number of non-histone substrates, including p53, E2F1, RelA, AR, and other important transcription factors. However, despite the ever growing list of potential substrates of Set7/9, the question of its substrate specificity is still debatable. To gain a better understanding of the Set7/9 substrate specificity and to clarify the importance of structural domains of Set7/9 for protein-protein interactions (PPIs) we determined interactomes for both MORN and SET domains of Set7/9 by pull-down assay coupled with mass-spectrometry. Importantly, we demonstrated that most of PPIs of Set7/9 are mediated via its MORN domain. The latter has preference towards positively charged amino acids that are often found in RNA- binding proteins. One of the Set7/ 9-interacting proteins was identified as Sam68, an RNA splicing protein with a KH (heterogeneous nuclear ribonucleoprotein K (hnRNP K) homology) domain. Importantly, the RG-rich domain of Sam68 that is also present in many splicing factors was found to interact with Set7/9. We revealed that Set7/9 not only co-immunoprecipitated with Sam68, but also methylated the latter on K208. Functionally, knockout of Set7/9 decreased the protein level of Sam68 in cells resulting in altered regulation of cell cycle and apoptosis. Finally, the bioinformatics analysis established a correlation between the high levels of Sam68/Set7/9 co-expression and better survival rates of patients with colon cancer. (C) 2020 Published by Elsevier