The Post-Transcriptional Regulation Mechanism and Functional Importance of a Key Maternal mRNA, GLP-1

Abstract

Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. C. elegans Notch homolog glp-1 maternal gene is necessary for germ cell proliferation, and embryonic fate determination. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3’ UTR. When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3’ UTR reporter transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of pos-1 increases the average polyA tail length of endogenous glp-1 transcripts in embryos. In this dissertation, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection. This result rules out alternative polyA site selection as a mechanism of regulation. We also show that wild-type glp-1 transgenic reporter embryos have a shorter average polyA tail length compared to transgenic reporters with mutated GLD-1 or POS-1 binding motifs. We have studied the effect of cytoplasmic polymerases, deadenylases and translation initiation factors on our transgenic reporters. Our RNAi experiments show that two cytoplasmic polyA polymerases, GLD-2 and GLD-4, have strikingly different effects on the expression of reporter transgenes harboring GLD-1 or POS-1 binding motif mutations. By contrast, none of the deadenylases affect the transgenic reporter expression. We also observed strong derepression of all reporters upon reduction of the translation initiation factor ife-3. The results reveal that POS-1 and GLD-1 exert their repressive effects in different ways through cytoplasmic polyA polymerase activity, while IFE-3 mediated translation repression is independent of both POS-1 and GLD-1. Lastly, we have examined the biological significance of glp-1 3’UTR to the worm reproduction by using CRISPR/Cas9 mutagenesis to generate glp-1 3’UTR mutations in the endogenous locus. Characterization of a 71 base pair mutation that deletes the GLD-1 and POS-1 binding sites in the glp-1 3’UTR reveals a 2-fold reduction in the number of embryos produced and a 4-fold reduction in the hatch rate. Imaging results show that the mutant embryos appear to have patterning defects. Together, our results show that the glp-1 3’UTR contributes to reproductive health but is not essential to fertility.