Apoptosis is a conserved fundamental biological cell death process critical for development, homeostasis, and stress responses. Dysregulation of apoptosis has profound effects on many diseases, including cancer. Recently, microRNAs (miRs) are found to modulate expression of many apoptotic proteins, and like apoptotic genes, miRs are often deregulated in human cancers. Recent evidence suggests complex combinatorial relationships between miRNAs and their targets. Thus, it will be important to establish an in vivo system to understand how miRs coordinate the expression of apoptotic genes in order to develop alternative therapies for cancer treatment. DNA damage in C. elegans germline activates p53-like protein CEP-1, which transcriptionally induces expression of BH3-only protein EGL-1 required to trigger apoptosis. Since the syncytial germline shares a common cytoplasm we hypothesize that mechanisms exist to buffer EGL-1 translation in surrounding cells with less damage. Inactivation of ALG-2/Argonaute as well as mir-35 and mir-71, predicted to bind the egl-1 3’ untranslated region (3’UTR), increase germ cell apoptosis in response to genotoxic stress. An in vivo fluorescent reporter with GFP fused histone H2B under control of germline-specific pie-1 promoter is used to assess egl-1 3’-UTR activity. pie-1 3’-UTR was used as a control since it lacks binding sites to targeted miRs in the egl-1 3’UTR. GFP intensity with egl-1 3’-UTR was much lower compared to the pie-1 3’-UTR. However, GFP expression with egl-1 3’UTR increased dramatically in mir-35 and mir-71 single mutants, suggesting these miRs limit EGL-1 translation. Ablation of mir-71 and mir-58 (also predicted to bind egl-1 3’-UTR) restricted GFP expression to germ cells and oocytes, whereas ablation of mir-35 restored GFP signal in germ cells, oocytes and embryos. This suggests specific miRs control EGL-1 translation in distinct regions of the germline. Interestingly, mir-58 deletion conferred apoptotic resistance and decline in GFP intensity, suggesting a pro-apoptotic role. mir-35 and mir-71 double mutant showed synergistic activation of apoptosis after genotoxic stress relative to single miR mutants. However, mir-35 and mir-58 double mutant showed ~50% reduction in cell death compared to mir-35 single mutant, supporting our observation that mir-58 positively regulates EGL-1. Our study highlights complex miRNA permutations under which EGL-1 is regulated. Understanding how miRs regulate pro-apoptotic proteins translation should illuminate conserved mechanisms that can be exploited for alternative cancer drug development.
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