CRK regulation of ribosome biogenesis and craniofacial development

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PROJECT SUMMARY/ABSTRACT Background and Overall Hypothesis: Ribosome biogenesis, the process by which the protein synthesizing machinery is produced, is essential and required in all cells. Mutations in factors required for ribosome production give rise to a subset of diseases called ribosomopathies. These diseases are surprisingly characterized by tissue-specific abnormalities despite the essential role of ribosomes in all cell types. Craniofacial specific defects, occurring in established ribosomopathies such as Treacher Collins syndrome, manifest from neural crest cell sensitivity to the nucleolar stress response during embryonic development. Miller-Dieker lissencephaly syndrome caused by microdeletion (17p13.3) also leads to craniofacial abnormalities, but with no defined molecular mechanism or connection to ribosome biogenesis thus far. These microdeletion phenotypes are further defined by inclusion of CRK resulting in more severe craniofacial defects. Accordingly, CRK’s role in development has been more precisely established with Crk (-/-) null mice exhibiting improper nasal development and cleft palate. CRK is a protooncogenic adapter protein that is present in several signaling pathways controlling cellular outputs such as proliferation and migration. Recently, the Baserga laboratory identified an additional role for CRK as a novel human ribosome biogenesis factor through a genome-wide siRNA screen in MCF10A breast epithelial cells (Farley-Barnes et al 2018). My preliminary results indicate that CRK depletion leads to inhibition of early pre-rRNA processing steps in ribosome biogenesis through controlling the stability of the essential U8 small nucleolar RNA (snoRNA). Furthermore, I have identified a promising CRK-mediated pathway for controlling U8 stability by U8 snoRNA trimethyl guanosine cap removal via the DCP2 decapping enzyme. I hypothesize that this newly identified role of CRK in ribosome biogenesis contributes to its importance in craniofacial development. Specific Aims: I propose to elucidate the molecular mechanisms underlying CRK’s control of human ribosome biogenesis in tissue culture cells. I will test for changes in U8 snoRNA and DCP2 protein localization and U8 snoRNA trimethyl guanosine cap abundance upon CRK depletion. Then I will test whether CRK directly interacts with the DCP2 containing decapping complex. Secondly, I will determine the extent to which CRK’s essential role in craniofacial development is connected to its role in ribosome biogenesis in the model organism Xenopus tropicalis. I will use morpholino oligos and CRISPR to deplete and knockout crk so that I can observe changes in embryonic morphology, neural crest development, pre-rRNA processing, and p53 nucleolar stress response induction. My proposed work will define CRK’s molecular role in craniofacial development through its connection to ribosome biogenesis for the first time. Additionally, my work will provide insight into the molecular basis of ribosomopathy tissue-specific defects and signaling control of pre-rRNA processing, areas that remain poorly understood.