Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network [expression]

In embryonic stem cell (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors that regulate the ESC state are not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 Ubiquitin Ligase Protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses revealed that MKRN1 is a novel RNA-binding protein that exists within messenger ribonucleoprotein (mRNP) complexes in undifferentiated ESC populations. In accordance with its presence in mRNPs, MKRN1 is mobilized to stress granules (SG) upon arsenite-induced stress, yet MKRN1 is not required for SG formation. RIP-chip analysis revealed that MKRN1 associates with mRNAs encoding functionally related regulatory proteins involved in diverse processes such as cell differentiation, apoptosis, or secreted proteins. Thus, our unbiased systems level analyses supports a role for MKRN1 as a novel RNA-binding protein and a potential gene regulatory protein within the ESC GRN. Expression profiling by microarray of mESC populations 6 days post transient transfection with pLKO.1 shGFP or pLKO.1 shMKRN1 plasmids. Transfected mESC populations were subjected to four days of puromycin selection to remove non-transfected cells prior to lysing the resultant puromycin-resistant populaton for RNA extraction. Differential gene expression in shMKRN1-transfected ESC populations was compared to shGFP-transfected control ESC populations.