Proteins of the PUF family play an important role in germ cell development in diverse organisms. These are RNA-binding proteins that function as translational regulators of their mRNA targets. Earlier results from our laboratory have shown that the C. elegans PUF protein, PUF-8, is essential for the maintenance of germline stem cells (GSCs). PUF-8, along with another RNA-binding protein called MEX-3, directly promotes GSC mitosis. To investigate the underlying mechanism, we disrupted cell cycle-related genes by RNAi on
puf-8(-) background and screened for suppression of any of the
puf-8(-) phenotypes. One of the phenotypes of
puf-8(-) is excessive production of sperm. This phenotype is suppressed by the RNAi-mediated disruption of
cyb-2.1, which encodes a cyclin B ortholog. As evidenced by the phenotypes of both
cyb-2.1(RNAi) and
cyb-2.1(
tm2027), a null allele, CYB-2.1 is required for neither GSC mitosis nor spermatogenesis. Consistently, CYB-2.1 does not appear to be produced in both mitotic and meiotic germ cells: transgenic worms carrying the GFP:
cyb-2.1 3'UTR transgene, do not express GFP in these cells. In contrast, removal of PUF-8 misexpresses GFP:
cyb-2.1 3'UTR in the mitotic germ cells, indicating that PUF-8 suppresses
cyb-2.1 translation in these cells via
cyb-2.1 3'UTR. Our gel shift assays reveal that the PUF-8 protein is capable of directly binding to
cyb-2.1 3'UTR. Further, mutations that abolish this binding misexpress GFP:
cyb-2.1 3'UTR in mitotic germ cells. These results show that PUF-8 suppresses
cyb-2.1 translation by interacting with its 3'UTR. GFP:
cyb-2.1 3'UTR transgene bearing 3'UTR mutations that abolish PUF-8 binding continue to suppress GFP expression in meiotic germ cells. We find that a different RNA-binding protein, namely GLD-1, suppresses
cyb-2.1 translation in the meiotic germ cells, again acting via
cyb-2.1 3'UTR, but through a sequence that is distinct from the PUF-8-binding sequence. Thus, our results show that both PUF-8 and GLD-1 control
cyb-2.1 translation sequentially - PUF-8 in the mitotic region and GLD-1 in the subsequent meiotic region - to prevent premature expression of CYB-2.1. These results support the model that the translation suppression is one of the mechanisms by which germ cells protect themselves from the influence of factors required later during embryogenesis.