Fig. 2. CAR-1 and CGH-1 associate together in a RNA-dependent complex. (A) Immunoprecipitation of endogenous CGH-1-associated proteins from one-day-old adult hermaphrodites, identified by mass spectroscopy. (B) Each CAR-1 ortholog includes a Sm-like domain within a characteristic conserved region (black) and a variable number of Arginine-Glycine-Glycine (RGG) triplets or functional equivalents (vertical bars) (Birney et al., 1993). An additional conserved region (shaded) contains a FDF domain, a sequence motif of unknown function (Anantharaman and Aravind, 2004). The percentage identity (Id) and similarity (Sim) to C. elegans CAR-1 within these regions was determined by pair-wise BLAST. CAR-1, C. elegans (NP_493254); Hs, H. sapiens (NP_056393); Tral, D. melanogaster (AAF49905); Scd6p, S. cerevisiae (NP_015454). (C) Interaction between CAR-1 and CGH-1 requires RNA. Co-immunoprecipitation of CAR-1 and CGH-1 from C. elegans extracts (lanes 2 and 6) was abolished by RNase A treatment (lanes 3 and 7). Lanes 4 and 8 show control incubations. (D) CAR-1 and CGH-1 are detected specifically in the germline. Lysates from one-day-old adult wild-type worms (N2) and germline-deficient glp-4(bn2) hermaphrodites (Beanan and Strome, 1992) were analyzed by western blotting with CAR-1 and CGH-1 antisera, using Tubulin as a control. The CAR-1 antibody recognized a single germline-specific species of 45 kDa, larger than the predicted size of 37.6 kDa. (E) Diminished CAR-1 protein levels in car-1(RNAi) worms. Protein extracts from wild-type (N2) or car-1(RNAi) one-day-old adults were analyzed by western blotting.