Post-Transcriptional Gene Expression of TNF? mRNA by an FXR1a-associated microRNP Significance MicroRNAs and RNA-protein complexes (RNPs) are post-transcriptional gene expression regulators that play essential roles in immunity and cancer. Quiescent (G0) cells are important in immunity and cancers, such as leukemia, where they resist clinical therapy and cause recurrences. G0 cells switch to a reversibly arrested state, maintained by distinct gene expression. G0 gene expression and mechanisms could provide new therapeutic options against resistant cancers. We revealed that in G0, the cytokine, TNF?, that promotes inflammation, tumorigenesis and G0, is translationally activated by an FXR1a-associated microRNP. Importantly, FXR1 increases in G0 and causes cell cycle arrest. FXR1a-microRNP, TNFalpha mRNA recruitment and translation mechanism remain to be characterized and would provide insights into G0 gene expression. Objective The primary goal of this study is to characterize the G0 FXR1a-microRNP in G0 leukemic cells that leads to recruitment and translation activation of TNF? mRNA. Specifically, this study will investigate FXR1a interactions that lead to TNF? mRNA recruitment by the microRNP for activation, elucidate the non-canonical translation mechanism of TNF? mRNA and characterize G0 microRNPs involved in TNF? mRNA activation. Premise MicroRNA-mediated activation requires a microRNP comprising the microRNP effector, AGO2, the RNA binding protein, FXR1a, and lacking the repressor, GW182. TNF? mRNA is recruited by the microRNP in the nucleus. During early G0 (<24hrs), repression is observed; activation is observed only in late G0 (>24hrs), indicating distinct early and late G0 microRNPs. In G0, canonical polyadenylated mRNA translation is reduced. Consistently, PARN deadenylase is required for translation of TNF? mRNA, which is deadenylated, and FXR1a interacts with translation factors, which promote non-canonical translation. Based on these data, we propose that specific mRNAs are directed for specialized translation by a distinct FXR1a-microRNP in late G0. Method First, the nuclear interactions of FXR1a with the microRNP that leads to TNF? mRNA recruitment in G0 monocytic leukemic cells will be characterized by overexpression, knockdown and complementation with mutants that affect interactions and activation, along with co-immunoprecipitations and functional analyses. Second, the mechanism of translation activation of TNF? mRNA will be elucidated by investigating the role of FXR1a interactions with translation factors and of PARN-mediated deadenylation in G0, using previously developed assays. Third, the early and late G0 microRNPs associated with TNF? mRNA in the nucleus and cytoplasm will be isolated by previously developed in vivo crosslinking coupled RNP affinity purification, to identify factors that regulate activation. Identified factors will be analyzed for their role in regulating activation. Outcome These functional studies of G0 microRNPs will provide a greater understanding of the role of RNPs in specific gene expression in G0, in particular, of critical cytokines, in clinically resistant leukemia cells.
MicroRNAs are powerful genetic materials that control the expression of critical cytokines that can lead to the development of several types of cancers and immune disorders and promote clinical resistance and cancer recurrences. Based on our previous studies, the primary objective of this project is to investigate the functions of microRNAs and their associated regulators in the expression of a critical inflammatory cytokine, TNF?, in quiescent monocytic leukemic cells, using our previously developed biochemical tools and techniques. An investigation of the regulated functions of microRNAs in cancer quiescence, which leads to chemoresistance and cancer resurgence, would provide significant insights into the aberrant expression underlying critical immune disorders, cancer resistance and persistence.
