The Pumilio proteins PUF-5 and PUF-6/7/10 are necessary for repression of Caenorhabditis elegans Notch/glp-1 during late oogenesis (or Not all that glitters is GLD-1)

In C. elegans, Notch/GLP-1 is vital to both germ cell and embryonic development. The glp-1 mRNA is translationally regulated in the germline and embryo to localize Notch signaling to specific cells at specific times of development. However, little is known about the factors that control glp-1 translation. To find genes that regulate glp-1, we used RNA interference to screen a library of genes shown by microarray analysis to have increased expression in the germline. From these germline-enriched genes, 97 were selected that encode potential RNA binding proteins. RNAi of these 97 genes identified a single candidate, puf-5, that represses GLP-1 expression in oocytes. PUF-5 protein is related to the Pumilio and FBF family of proteins, several of which control mRNA translation or stability in various organisms. Following puf-5 RNAi, GLP-1 expression was de-repressed in late-stage oocytes from early diakinesis to immediately before oocyte maturation. In other regions of puf-5(RNAi) germlines and embryos, GLP-1 regulation was largely unaffected. In addition, we found that three nearly identical PUF proteins, PUF-6, PUF-7, and PUF-10, but not other PUF proteins, also were required for glp-1 repression in oocytes. Loss of PUF-5 or PUF-6/7/10 also caused defects in oogenesis. Furthermore, PUF-5 protein was detected in late stage oocytes by immunofluorescence, but was not detectable in early stage germ cells of the distal gonad arm. Recombinant PUF-5 and PUF-6 bound weakly but specifically to a small region of the glp-1 3' UTR in vitro. However, it is not yet known if direct binding to glp-1 mRNA is important for translational control in vivo. These studies, together with previous work, suggest that distinct RNA binding complexes repress maternal mRNA translation at different stages of germ cell development. The KH protein GLD-1 represses several mRNAs including glp-1 during early oogenesis, while PUF-5, PUF-6, PUF-7, and PUF-10 are key components of a regulatory system that represses Notch/glp-1 and probably other maternal mRNAs during late oogenesis.