A genetic screen identifies C. elegans eif-3.H and hrpr-1 as pro-apoptotic genes and potential activators of egl-1 expression

Abstract

During C. elegans development, 1090 somatic cells are generated of which 131 reproducibly die, many through apoptosis. The C. elegans BH3-only gene egl-1 is the key activator of apoptosis in somatic tissues, and it is predominantly expressed in 'cell death' lineages i.e. lineages in which apoptotic cell death occurs. egl-1 expression is regulated at the transcriptional and post-transcriptional level. For example, we previously showed that the miR-35 and miR-58 families of miRNAs repress egl-1 expression in mothers of 'unwanted' cells by binding to the 3' UTR of egl-1 mRNA, thereby increasing egl-1 mRNA turnover. In a screen for RNA-binding proteins with a role in the post-transcriptional control of egl-1 expression, we identified EIF-3.H (ortholog of human eIF3H) and HRPR-1 (ortholog human hnRNP R/Q) as potential activators of egl-1 expression. In addition, we demonstrate that the knockdown of the eif-3.H or hrpr-1 gene by RNA-mediated interference (RNAi) results in the inappropriate survival of unwanted cells during C. elegans development. Our study provides novel insight into how egl-1 expression is controlled to cause the reproducible pattern of cell death observed during C. elegans development.

Figure 1. Genetic screen for activators of egl-1 expression using RNA-mediated interference

A) Flowchart of genetic screens for activators of egl-1 expression. (B) Primary (positive) screen for activators of egl-1 expression. After RNAi-mediated knockdown of RBP genes, candidates were identified by screening for a decrease in GFP:: HIS-58 signal in oocytes of animals carrying the reporter P _{mai- 2} gfp:: his-58 :: egl-1 3′ UTR ( bcSi26 ). White arrows point to GFP:: HIS-58 signal in oocyte nuclei. (C) Secondary (negative) screen. Nonspecific candidates were eliminated by screening for a decrease in GFP:: HIS-58 signal in embryos of animals carrying the reporter P _{mai- 2} gfp:: his-58 :: mai-2 3′ UTR ( bcSi25 ). (D) NSM sister cell (NSMsc) survival screen. (Top) Schematics showing the NSM lineage in wild-type ( +/+ ) and ced-3 (717) animals (Ellis and Horvitz, 1986). The two bilaterally symmetric neurosecretory motoneuron (NSM) neuroblasts (NSMnb) (left and right) divide ~410 minutes after the first zygotic cleavage (at 20°C), each generating one NSM neuron, which is programmed to survive, and one NSM sister cell (NSMsc), which is programmed to die (‘unwanted' daughter cell) (Sulston et al., 1983). In wild-type ( +/+ ) animals, the NSMsc undergoes apoptotic cell death, resulting in one NSM from each NSM neuroblast. When apoptosis is blocked, the NSMsc inappropriately survives, resulting in an extra ‘NSM-like' cell. The NSM and the ‘undead' NSMsc can be identified in the anterior pharynx of L3/L4 larvae using the reporter P _tph-1 gfp:: his-24 (Yan et al., 2013). (Bottom) RNAi knockdown of eif-3.H or hrpr-1 causes NSMsc survival. To enhance RNAi efficiency in the NSM lineage, RNAi experiments were also performed in the nre-1 ( hd20 ) lin-15b ( hd126 ) background (Schmitz et al., 2007). The percentage of NSMsc survival is enhanced in the background of n2427 , a weak loss of function mutation of ced-3 (Shaham et al., 1999). The sample size (n) is shown in the table. The complete genotypes of strains used are provided in Table 1.