Programmed cell death is an important process in animal development and requires strict control to avoid the unwanted death of cells. In C. elegans, the pro-apoptotic BH3-only gene egl-1 is the most upstream factor of the core apoptotic pathway, and its activity is thought to determine the life-versus-death decision during development. Previous studies have identified regulatory modules that control the transcription of egl-1 in a lineage-specific manner; however, investigations into the post-transcriptional regulation of egl-1 are largely absent. We find that the mir-35 family and mir-58 bantam family of miRNAs directly target the 3'UTR of egl-1 mRNA and act cooperatively to suppress its expression. This suppression affects both mRNA copy number and translation efficiency, and is crucial to prevent the precocious and collateral death of mothers and sisters of cells programmed to die. Using single-molecule RNA FISH, we show that egl-1 is already transcribed in mothers of cells programmed to die and that mir-35 family and mir-58 bantam family miRNAs prevent their precocious death by keeping the copy number of egl-1 mRNA below a critical threshold. These miRNAs also act to dampen the transcriptional boost of egl-1 that occurs specifically in the daughter that is programmed to die, but they are generally not required for the degradation of egl-1 mRNA in the daughter that is programmed to survive. We propose that these miRNAs function to adjust lineage-specific differences in egl-1 transcriptional activation and thereby ensure that egl-1 expression reaches a level sufficient to trigger death only in the daughters that are programmed to die. Finally, we investigate the role of PUF (Pumilio/FBF) RNA-binding proteins (RBPs) in the post-transcriptional regulation of egl-1, whose 3'UTR contains two FBF binding elements. Mutations in these elements mediate increased reporter expression in both the germline and early embryos, suggesting that egl-1 is directly regulated by FBF during development. Although miRNAs and RBPs can function independently, recent studies have found these two pathways can interact in the regulation of shared targets, and we speculate that egl-1 may be one such target.

Affiliations:
- Center for Integrated Protein Science Munich - CIPSM, Department Biology II, Ludwig-Maximilians-University Munich, Planegg-Martinsried 82152, Germany
- Program in Molecular Medicine, RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01606, USA