Functional characterization of the mRNA-binding protein ZC3HAV1 in stem cell renewal and differentiation

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RNA-binding proteins bind cis-regulatory elements on transcripts to orchestrate mRNA biogenesis, translation and turnover. The detailed functions of RBPs in embryonic stem cells and their dynamic changes in binding activity during differentiation have yet to be addressed. To this end, we applied mRNA interactome capture of pluripotent mouse embryonic stem cells and their differentiation to neurons to identify differentially binding RBPs. The mRNA binding protein ZC3HAV1 showed transient changes in mRNA-binding during the course of differentiation. ZC3HAV has been mostly studied as an antiviral factor involved in decay and translation of viral transcripts. We will deliver three major milestones that are independent from each other, but cumulate into a system-wide description of ZC3HAV1 specificity and regulation in stem cell self-renewal and differentiation, providing the first description of ZC3HAV1 function on cellular mRNA target transcripts. We will map the mRNA interactome of ZC3HAV1 using protein-RNA crosslinking, immunoprecipitation, and next-generation sequencing to identify mRNAs where ZC3HAV1 binds, the region within the mRNA, and how the repertoire of putative targets changes. We will characterize the repertoire of protein interaction partners and ZC3HAV1-protein complexes through proximity biotinylation followed by mass spectrometry. To characterize the global molecular impact of ZC3HAV1 function, we will deplete and overexpress RNA-binding protein and determine changes in mRNA half-lives and ribosome occupancy. These three assays provide an overview of the changes that putative target transcripts may undergo with respect to their mRNA stability and translation. In addition, we will examine the role of ZC3HAV1 during cell differentiation.