Stem cell research is revolutionizing the way scientists think about mammalian development. It is a field full of promise to treat many debilitating and degenerative diseases. Regulation of stem cells by transcription factors that bind to specific DNA loci to control gene regulatory networks have been well studied in various stem cell populations. However, the mechanisms by which the regulation of post-transcriptional RNA processing influences the timely, highly specialized differentiation of stem cells are not well understood. Only recently have researchers started focusing attention on the importance of RNA regulation in the control of gene expression and, thus, to impact human health. RNA binding proteins (RBPs) bind to and regulate RNA metabolism at multiple levels from processing within the nucleus, nuclear export, and transport to organelles within the cytoplasm. In addition, RBPs can regulate RNA stability and protein translation. Techniques to study and identify the RNA substrates regulated by RNA binding proteins have only recently been developed within the last decade, with the availability of high-throughput sequencing revolutionizing established techniques, enabling transcriptome-wide views of RNA biology. My dissertation research focuses on the function of the IGF2BP/IMP family of RNA binding proteins. In particular, I have investigated the role of IGF2BP1/IMP1, in early human development using human embryonic stem cells (hESCs) as a model system. I have used CLIP-seq to identify the endogenous targets of IMP1 in hESCs and have determined the mechanism of regulation for the novel target ITGB5. Additionally, I have uncovered a role for IMP1 in early neural progenitor cell populations and in localization of target RNAs. Ongoing studies comparing and contrasting the various targets and functions of IMP1 and IMP2 in hESCs at the genome-wide level will pave the way for future investigators to dissect the individual functions for each of these RBPs. This work will provide a resource for those aiming to understand the mechanisms of how these proteins control their target RNAs during development and tumorigenesis

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.