miRNAs play critical roles in development and other cellular processes in C. elegans even though most individual miRNAs are not essential for development or viability (Miska et al. PLos Genetics 2007). Extensive studies in the field have suggested that most miRNA functions are executed through complex miRNA-target interaction networks that function with other regulatory systems to shape gene expression dynamics for proper physiological functions. Hypothesizing that miRNAs collaborate with other gene regulation mechanisms to maintain C. elegans developmental robustness, we carried out a genome wide RNAi screen for genes that interact with the miRNA induced silencing complex (miRISC). We found that when the miRISC is compromised by knocking out either ain-1 or ain-2, two partially redundant GW182 proteins, many genes become essential for development (Weaver, Zabinsky et al. eLIFE 2014). One such gene is a conserved RNA binding protein that has not been studied in C. elegans. In an ain-2(lf) background, RNAi of the RNA binding protein causes 90% L1 larval lethality, whereas the lethality is not associated with either ain-2(lf) or RNAi alone. By using the CRISPR/Cas9 system to knock out this gene, we observed that 10% of progeny failed to develop past the L1 larval stage. However, in combination with ain-2(lf), the newly generated knock out exhibited nearly 100% L1 larval lethality, suggesting these two genes function redundantly to regulate post-embryonic development.
We hypothesize that this RNA binding protein may function with miRNAs to repress some of the same target mRNAs to regulate development. To identify specific miRNAs involved in this interaction, we carried out another screen and identified five miRNAs that exhibit a partial synthetic lethal phenotype with the RNA binding protein. These results revealed previously unknown functions of these miRNAs in post-embryonic development and developmental robustness. We hypothesized that the predicted targets of these miRNAs that function in the intestinal miRISC may also be regulated by the RNA binding protein since expression of ain-2 in the intestine partially suppresses the double mutant phenotype. We compared the predicted miRNA targets to previous data from our lab identifying intestinal mRNAs associated with the miRISC (Kudlow et al. Molecular Cell 2011) to find 54 likely miRNA targets. Further qRT-PCR analysis will determine how many of these candidates are common targets repressed by the newly identified RNA binding protein and specific miRNAs. Our results may shed light on the mechanism of how miRNAs work with RNA binding proteins to regulate gene expression for robust development.
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