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.