P granules are distinctive ribonucleoprotein complexes observed specifically in the cytoplasm of germ cells throughout development. We previously identified PGL-1 as being a constitutive protein component of P granules. The presence of an RGG box predicts that PGL-1 is an RNA-binding component of P granules.
pgl-1 mutants contain defective P granules and are sterile, due to defects in proliferation and gametogenesis. Interestingly, the sterility caused by null alleles of
pgl-1 is highly sensitive to temperature. Our identification and analysis of two additional
pgl-1 -related genes, termed
pgl-2 and
pgl-3 , demonstrate that the PGL proteins function redundantly, and at low temperature, PGL-2 and PGL-3 are sufficient for fertility. PGL-2 has 34% identity with PGL-1 in its N-terminal region. PGL-3 has 62% identity with PGL-1 throughout its length and contains an RGG box. Based on yeast two-hybrid and GST pull-down results, the three PGL proteins interact with each other. Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from both embryo and oocyte extracts, indicating that they are indeed in the same protein complex in vivo . Immunofluorescence analysis has demonstrated that in wild-type worms PGL-3 is associated with P granules at most stages of development, like PGL-1, but interestingly, PGL-2 is associated with P granules only during postembryonic development. Based on molecular epistasis results, each PGL protein associates with P granules independently of the other two. To address whether PGL-3 functions redundantly with PGL-1, we isolated a
pgl-3 deletion allele. We found that
pgl-1;
pgl-3 double mutant hermaphrodites and males show significantly enhanced (but not 100%) sterility at low temperature, compared to either single mutant. Double mutant hermaphrodites contain a severely underproliferated germline, indicating that the primary defect is in proliferation. Depletion of
pgl-2 by RNAi did not enhance sterility further. O ur findings suggest that both PGL-2 and PGL-3 are components of P granules that interact with PGL-1 and that at least PGL-3 functions redundantly with PGL-1 to ensure fertility in both sexes during C. elegans germline development.