RNA binding proteins control the molecular fate of RNA substrates and are often underappreciated regulatory factors in development. In recent work, we identified the RNA binding protein MSI2 in the control of human hematopoietic stem cell self-renewal (Rentas et al, Nature, 2016) and establish RNA binding proteins as key regulators of this process. In this proposal, we aim to functionally characterize using gold-standard in vitro and in vivo assays a novel MSI2-interactor and putative HSC-regulator. We will also utilize a combination of mass spectrometry and cytometry (single-cell) to identify MSI2-interacting RNA binding proteins in primitive hematopoietic cells. We will apply our enhanced CLIP-seq approach to identify RNA substrates of these RBPs and identify critical pathways which we can manipulate to enhance HSC self-renewal or direct HSC fate. We will utilize cutting edge methods like our recently published RNA-targeting Cas9 (Nelles et al, Cell, 2016) to identify novel RBPs and post-transcriptional circuitries essential for HSC function.
In this proposal, we will utilize cutting edge proteomics and custom-designed functional screens to identify novel RNA binding proteins important for hematopoietic stem cells (HSC) fate, and characterize their targets by enhanced CLIP-seq and other genomics methods. If successful, these insights will provide fundamental insights into regulation of hematopoiesis, supporting translational research in leukemia and other diseases of the hematopoietic system.
