Thursday, April 25, 2024
4/25/2024
PROJECT SUMMARY Survival and proliferation are fundamental cell activities that are exploited during oncogenesis. A key cellular pathway that supports survival and proliferation is the nuclear factor-Kappa B (NF-¿B) signaling pathway, which is also hyperactive in cancer cells and is a noted element that contributes to therapeutic resistance, a phenomenon observed in various cancers, including acute lymphoblastic leukemia (ALL). With an incidence of about 30 cases per million, ALL is the most common cancer among children and the most frequent cause of death from cancer before 20 years of age. In a recent study, it was shown that the elevated expression of coilin, the Cajal body (CB) marker protein, is a negative prognostic factor in ALL patients. Recent work by our lab has discovered that coilin is a positive regulator in the biogenesis of microRNAs (miRNAs), a class of noncoding RNAs that negatively regulate gene expression. However, the mechanism for this function is unclear. Another positive regulator of miRNA biogenesis is N6-methyladenosine (m6A) RNA methylation, a reversible modification that is installed by writers, removed by erasers, and acted on by readers. Our lab hypothesized that coilin was influencing miRNA biogenesis through the regulation of m6A RNA methylation and we have generated exciting preliminary data showing that coilin suppression results in a decrease in the expression of m6A writer METTL3, reader hnRNPA2B1, and eraser ALKBH5 proteins, as well as a their mislocalization within the cell and a reduction in global m6A RNA levels. Additionally, work published by our lab found that coilin positively regulates the expression of miR-517, a NF-¿B activator. Since the NF-¿B pathway is both regulated by and a regulator of the biogenesis of several miRNAs, and in consideration of previously published work on coilin, we have generated the central hypothesis that coilin supports m6A RNA methylation through which it regulates miRNA biogenesis and facilitates the survival and proliferation of the cell. This hypothesis will be tested with the use of various primary and transformed cell lines, and our newly generated, CRISPR/Cas9-engineered coilin zebrafish models. To test our central hypothesis, two specific aims have been generated for which we will test the hypotheses that: 1) Coilin directs the optimal synthesis, posttranslational modification, and localization of m6A RNA Writers, Readers, and Erasers; and 2) Coilin mediates m6A RNA methylation by which miRNA biogenesis and survival & proliferative pathways are regulated. This work will characterize three novel CRISPR-engineered zebrafish coilin lines while also investigating a novel pathway by which coilin mediates m6A RNA methylation and regulates miRNA biogenesis. Findings in this proposal will highlight a novel role for coilin in facilitating survival and proliferation, as well as a potential prognostic or therapeutic role for coilin in cancers, such as ALL.
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