The Role of the RING Finger Protein Makorin-1 in the Embryonic Stem Cell Gene Regulatory Network

Stem cell behaviour is orchestrated as a balance of molecular events that ultimately activate and repress gene expression. These molecular events are collectively referred to as the gene regulatory network (GRN). In embryonic stem cells (ESCs), GRNs coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors that regulate these processes is not fully understood. Our previous temporal microarray analysis of undifferentiated and early committed mouse ESCs identified Mkrn1 as a gene that is preferentially expressed in undifferentiated ESCs and concomitantly down-regulated early upon commitment. Using multilayered systems-level analysis we explored the molecular and cellular functions for the RING Finger Protein Makorin-1 (MKRN1) in undifferentiated ESC populations. Specifically, using a FLAG:MKRN1 ESC clone we compiled an MKRN1-centered `interactome' from undifferentiated ESC populations at the proteomic and ribonomic level. We find that in undifferentiated ESC populations, MKRN1 is predominantly associated with proteins involved in mRNA metabolism including regulators of mRNA turnover, transport and/or translation. At the RNA level, FLAG:MKRN1 associates with a varied set of transcripts encoding for functionally related regulatory proteins involved in processes such as cell differentiation, apoptosis, and/or mRNAs encoding proteins that are destined for translation through the secretory pathway. Informed by our integrated network analysis, we found that MKRN1 is a novel stress granule resident protein, which prompted us to explore MKRN1 function in ESC populations downstream from the oxidative and genotoxic stress responses. Manipulation of MKRN1 expression in ESC populations undergoing cellular stress resulted in modest increases in early apoptotic markers depending on the type of stress response evoked, but manipulation of MKRN1 was not found to promote or impair the extent of nuclear fragmentation (a hallmark of apoptosis) in ESC populations responding to oxidative or genotoxic stress. Our unbiased systems level analysis of MKRN1 function in undifferentiated ESC populations supports a role for MKRN1 as a messenger ribonucleoprotein in ESCs and a potential gene regulatory protein within the ESC GRN.