Figure 5. Differential GLD-4 and GLD-2 expression in the proliferative zone is FBF dependent. (A) GLD-4 expression is equal across the distal germ line. GLD-2 intensities increase from low-to-high in a distal-to-proximal manner. Extruded gonads of indicated genotype stained with DAPI, a-GLD-2, a-GLD-4, and a-GLH-2 as a positive tissue penetration control (not shown). Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. (B,C) Distal GLD-2 expression is repressed by fbf activity. (B) Example of an fbf(RNAi) immunostained extruded gonad. For the complete RNAi experiment see Figure S1. (C) Quantification of the complete fbf(RNAi) experiment. Four different regions of nine germ lines per genotype were analyzed in their median, primarily cytoplasmic area. Error bars are SEM. ***, p,0.001; **, p,0.01; *, p,0.05; bars without indicated p value are statistically not significant (Student's t-test). (D, E) FBF binds specifically to at least one of the five predicted sequence elements in the
gld-2 39UTR. (D) Schematic drawing of the 1094 nt long
gld-2 39UTR. Sequence alignment of FBF-binding element consensus (FBE cons.) sequence [14] and the conserved FBE4 element in three Caenorhabditis species: ce, C. elegans; cb, C. briggsae; cr, C. remanei. pA indicates beginning of the poly(A) tail. (E) Yeast three-hybrid assay. RNA hybrid and Gal4-protein fusions are indicated. FBF-1, FBF-2 and PUF-5 belong to same RNA-binding protein family. Note, the wild-type (wt) and mutant (mut) sequence of FBE4 tested is larger than the given sequences (see Materials and Methods). A positive and negative control RNA was included (not shown) and protein expression was confirmed by western blotting (not shown). (F) LAP-tagged FBF-2 associates with endogenous
gld-2 mRNA in RNA-coimmunoprecipitation experiments (RIPs) directed against the GFP portion of the fusion protein.