mTOR-regulated U2AF plasticity and alternative polyadenylation

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mTOR-regulated U2AF plasticity and alternative polyadenylation PROJECT SUMMARY/ABSTRACT U2AF (U2 auxiliary factor, comprised of U2AF1 and U2AF2) is an essential splicing factor and functions in 3’splice site selection during pre-mRNA processing. U2AF has been known to form a constitutive heterodimer and is important for alternative and constitutive splicing. However, preliminary data show that mTOR (mammalian target of rapamycin) signaling pathway controls the U2AF heterodimerization by U2AF2 phosphorylation- dependent manner. These regulated interactions between U2AF1 and U2AF2 constitute the U2AF plasticity. This newly discovered U2AF plasticity is a key element in alternative splicing and alternative polyadenylation. Based on these findings, the following central hypothesis can be proposed: the U2AF plasticity is a gateway to mTOR-regulated transcriptome reprogramming. The goals of this proposal are to investigate the regulatory mechanism of U2AF plasticity by mTOR and understand how this U2AF plasticity programs the transcriptome by focusing on alternative splicing and alternative polyadenylation. To this end, two specific aims are proposed. In the first aim, the role of mTOR-U2AF plasticity in transcriptome reprogramming will be investigated. CRISPR/Cas9-mediated genome engineering will be conducted to build up cell models which will constitutively polarize the U2AF plasticity in one way or the other. These cell models will be then tested for cell phenotypic changes and the transcriptomic changes will be profiled. Mutations in U2AF1 are prognostic in acute myeloid leukemia and myeloid dysplasia. Physiological relevance of these mutations to U2AF plasticity will be tested and a current model for disease pathogenesis will be challenged. For these tasks, a new bioinformatic pipeline will be developed. In the second aim, the regulatory axis that connects mTOR, U2AF plasticity, and histone biogenesis will be dissected. A kinase(s) that controls the U2AF plasticity will be identified. Also, the mechanism by which the U2AF plasticity programs alternative splicing and alternative polyadenylation will be delineated. Finally, the outcome of mTOR-U2AF plasticity-mediated alternative polyadenylation in the histone biogenesis will be examined. Together, this project will advance the understanding of transcriptome programming by mTOR- coordinated U2AF plasticity and suggest mechanistic cascades that communicate extracellular/cellular environments to gene expression programs. It will also challenge a current model of U2AF1 mutations in cancer pathogenesis. Moreover, this project will establish a link between mTOR and histone biogenesis through U2AF plasticity.