The translation regulation of pro-apoptotic genes

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PROJECT SUMMARY (PARENTAL AWARD) Inactivation of the Retinoblastoma 1 protein (pRB) is found in a substantial fraction of human cancers. This is because pRB inhibits the activity of three important E2F transcription factors (E2F1-3) that control the expression of numerous tumor-promoting genes, which control cell growth including cell cycle genes and metabolic factors. Conversely, the E2Fs also regulate the transcription of tumor-inhibiting processes including important apoptosis and necrosis genes. This Rb-controlled coupled expression of proliferation and cell death genes is designed to prevent uncontrolled cell growth that is a hallmark of cancer. However, the widespread loss of pRB in cancer underlines a major unsolved puzzle: why don’t pRB-deficient cells simply die? To answer this question, we profiled RNA and protein changes following pRB-depletion and found that essential cell death genes were indeed transcribed, but the mRNAs were not translated into functional proteins. We then searched for RNA-binding proteins (RBPs) that bound to and blocked the Ribosome occupancy of these cell- death genes. This analysis identified the PUMILIO (PUM) family of RBPs as potential regulators of cell death RNAs. Importantly, as would be predicted by such a regulatory role, co-depletion of PUM1 and PUM2 resulted in the apoptosis of RB1-deficient cancer cells. However, the high level of functional redundancy has limited functional dissection of PUM1 and PUM2 mechanics in specific translational regulation of cell death mRNAs. To circumvent this technical issue, we have engineered PUM1-/-, PUM2-/- CRISPR-derived knockout human cells that additionally express individually degradable PUM proteins. Following the addition of the plant hormone, AUXIN, PUM1 or PUM2 are rapidly degraded, enabling kinetic analysis of the stability, localization and translation initiation of Rb-regulated mRNA transcripts. Using well-defined candidate cell death genes, we propose to determine the mechanism(s) associated with mRNA regulation by the individual PUM1 or PUM2 proteins. We will then determine which PUM regulatory mechanisms are required in the development of RB1-/- Small Cell Lung Cancer (SCLC) in vivo.