Regulation of mRNA processing: Mechanisms and Consequences

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Synthesis of eukaryotic mRNAs is a complex process, and includes splicing and 3’ end formation of mRNA precursors. My lab has studied these processes for many years, including recently how they function in differentiation and disease. This proposal continues our studies on these topics, and can be divided into the following areas. mRNA processing in cancer. Work investigating how mutations in genes encoding splicing factors lead to MDS and other neoplasms will be continued, with an emphasis on SF3B1. Studies to elucidate both the mechanism(s) by which SF3B1 mutations affect splicing as well as the pathways that are dysregulated and lead to disease will be pursued. With respect to mechanism, an immediate goal will be to continue characterizing the SF3B1-interacting region of SUGP1. Another goal is to identify the SUGP1- interacting RNA helicase and determine its function in normal BP recognition, which will then allow determination of the detailed mechanism by which SF3B1 mutations disrupt splicing. With respect to pathways, experiments to elucidate the details of the recently described MAP3K7/p38/GATA1 pathway that underlies anemia in MDS will be performed. Missplicing events that affect other relevant pathways, such as aberrant activation of NF-kB, will be studied. FUS and other RBPs in ALS/FTD. With respect to FUS, experiments to analyze FUS nucleocytoplasmic homeostasis, a process important for formation of toxic cytoplasmic FUS aggregates, will be continued. Evidence supporting a gating mechanism involving the NPC and interactions with specific nucleoporins, themselves implicated in amyotrophic lateral sclerosis (ALS), will be pursued. Suggestions that this process may involve cell-type specificity, with MNs perhaps being more permissive, will be investigated. Experiments pursing the observation that RBP aggregates and consequent missplicing occur in a large fraction of sporadic ALS/FTD patient brains in the absence of known mutations will be continued. Aggregates will be isolated from motor cortex samples and protein/RNA composition determined to investigate what might nucleate their formation. Regulation of PA factor activity by AS. Experiments investigating the functions of isoforms of the polyadenylation (PA) factor WDR33 will be pursued. Two short isoforms, v2 and v3, are produced by intronic PA), and v2 but not v3 is an inner nuclear membrane protein not directly involved in PA. Interacting proteins of both will be identified, and results suggesting they are upregulated by an NF-kB pathway will be pursued. Evidence that the two isoforms function in the antiviral response, including to SARS-CoV-2 infection, will be further investigated. The finding that 45 isoforms of the PA factor Fip1 are produced in humans will be explored. eRNAs as mRNAs. Results suggesting that certain nuclear unstable lncRNAs, including eRNAs, are stabilized, exported to the cytoplasm and in some case translated, will be pursued. Observations that up to 5% of eRNAs contain significant ORFs and may be translated when the exosome cofactor Mtr4 is depleted will be further investigated. The possible physiological significance of eRNA “activation” will be studied in differentiated hESCs, which were shown to have sharply reduced levels of Mtr4.