Jeongsik Yong, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Most, if not all, cancer cells proliferate much faster than normal cells. Thus, studying how cancer cells proliferate faster than normal cells is a key in understanding cancer biology. The mammalian target of rapamycin (mTOR) pathway is a cellular pathway that controls cell proliferation and this pathway is commonly dysregulated in many cancers. Therefore, understanding the role of mTOR pathway in cell proliferation is important. When cells are activated to proliferate, the first thing they do is producing a lot of proteins. To make more proteins in cells, they need to make more messenger RNAs (mRNAs) from DNA. The whole procedure is called gene expression and mRNA is a key molecule in this procedure. Thus, the questions of how mRNAs are made and how they are regulated in cancer mechanisms are important questions to ask to understand cancer at a molecular level. Generally, mRNA undergoes very complicated process to make it competent for protein synthesis in cells. Recently, we discovered a pervasive production of truncated mRNAs when mTOR is activated in cells. The truncated mRNAs are produced by dysregulation of one of the steps during mRNA synthesis in cells. The cellular consequence of this phenomenon is the production of truncated proteins. Usually, fundamental elements of many proteins are consisted of catalytically active domains and regulatory domains. The active domain represents the function of a protein and the regulatory domain is a platform for fine-tuning of the protein activiy regulated by other cellular proteins. Interestingly, many truncated proteins produced by mTOR activation were lacking the regulatory or catalytic domain. This suggests that mTOR activation produces many deregulated super isoform proteins by truncation and this could be a driver to fast cell proliferation and cancer initiation at a molecular level. Based on this, we hypothesize that the same phenomena happen when cancer cells are activated to proliferate by mTOR. We searched cancer databases and found numerous candidate mRNAs for truncation in cancer, which was not recognized previously. Our goals in this proposal are to find them and understand their function in cancer cell proliferation using a series of experiments employing high profiling technologies including next generation sequencing and multi-dimensional LC-MS/MS. More importantly, we will narrow down the list of cancer-specific truncated mRNAs and finalize the critical truncated mRNAs by validating their existence in cancer patient database. The identified cancer-specific truncated mRNAs will be new targets in cancer research and provide novel platforms for the development of multiple biomarkers at both protein and RNA levels.

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.