During Caenorhabditis elegans (C. elegans) development, 1090 somatic cells are generated of which 131 reproducibly die, mostly through apoptosis. Apoptosis during C. elegans development is dependent on the conserved central apoptosis pathway, whose most upstream component is the pro-apoptotic BH3-only gene egl-1. Its overexpression can cause ectopic apoptosis. Unlike the other components of the central apoptosis pathway (i.e. ced-9 BCL-2, ced-4 Apaf-1, and ced-3 caspase), which are broadly expressed, egl-1 is predominantly expressed in ‘cell death lineages’. It was previously demonstrated that egl-1 BH3-only transcription is controlled by transcription factors in a ‘lineage-specific’ manner and initiated in mothers of cells programmed to die. After mother cell division, the number of egl-1 transcripts increases in the daughter that is programmed to die and decreases in the daughter that survives. In addition, post-transcriptional regulation has been demonstrated to play a critical role in fine-tuning egl-1 expression. For example, the miR-35 and miR-58 families of microRNAs repress egl-1 expression in the mother of cells programmed to die by binding to their binding sites located in the egl-1 3′ UTR. The loss of these microRNAs causes precocious mother cell deaths due to the up-regulation of egl-1 expression in mother cells.
Considering the critical roles of 3′ UTRs of mRNAs in post-transcriptional gene regulation, this study first investigates the sequence features of the egl-1 3′ UTR. In addition to the binding elements for miR-35 and miR-58 microRNAs, this study identifies other elements that are evolutionarily conserved among species, including four FBF-binding elements (FBEs) and a 3′ terminal element (TPTE). Mutation in any of these cis-acting elements leads to de-repression of the egl-1 3′ UTR reporter (Pmai-2gfp::h2b::egl-1 3′ UTR) whose expression is under the control of the egl-1 3′ UTR. Besides, the disruption of the TPTE in the endogenous egl-1 gene causes the appearance of large cell corpses during embryo development, which is indicative of precocious mother cell death or ectopic cell death. These results indicate that the cis-acting elements in the egl-1 3′ UTR contribute to the modulation of egl-1 expression.
Additionally, I perform a genetic screen, in which 660 genes predicted to encode RNA-binding proteins (RBPs) are individually knocked down by RNA interference (RNAi), to identify RBPs that could be involved in the regulation of egl-1 expression. Through this screen, five repressor candidates and two activator candidates of egl-1 were identified. The loss of the repressor candidates up-regulates the expression of the egl-1 3′ UTR reporter (Pmai-2gfp::h2b::egl-1 3′ UTR) and results in the appearance of large cell corpses, which are indicative of precocious/ectopic cell death. The loss of activator candidates down-regulates the expression of the egl-1 3′ UTR reporter (Pmai-2gfp::h2b::egl-1 3′ UTR) and causes the survival of cells that normally die via apoptosis.
Finally, the present study describes the development of a novel method, based on the SunTag system, for the live imaging of the translation of an individual egl-1 mRNA in real time. The SunTag system amplifies signals from the protein of interest by recruiting multiple copies of the fluorescent protein to a polypeptide scaffold that is fused to the protein of interest. Taking advantage of this method, egl-1 mRNA translation is visualized with a high sensitivity, and the potential spatiotemporal expression pattern of egl-1 in specific lineages where apoptotic cell death occurs is investigated.
In summary, the data presented in this work contributes to our understanding of the control of egl-1 expression and apoptosis. In addition, the development of a method to visualize egl-1 mRNA translation in vivo in developing C. elegans enables future research on the control of the spatiotemporal expression of egl-1 BH3-only gene.