Fig. 5. The Ras-MAPK pathway functions with PUF-5 to repress endogenous glp-1 mRNA.

(A) Dissected gonads were stained by immunoflorescence with α-GLP-1. Only the proximal part of gonads is shown. Gonads are outlined by thick white lines, oocytes by thin white lines. Gonads were stained in parallel from the same experiment with matched exposure times and adjustments. Insets: GLP-1 staining in the tip of the distal gonad was used as an internal control. Scale bare: 50 μm. (B) GLP-1 protein expression was quantified by measuring GLP-1 pixel intensity mean in a 50 μm diameter circle of the most intense proximal oocyte and normalized to the distal tip. Strains were N2 or mpk-1(ga111ts), with empty vector RNAi (mock) or puf-5(RNAi) (puf-5) (n=8-24 for each strain/RNAi). Error bars represent s.d. for one experiment. Similar results were obtained with three independent experiments. (C) Gonads after the indicated single or double RNAi were dissected and GFP levels of the glp-1 3’UTR reporter were measured as in Fig. 4 (n=10-18 for each strain/RNAi). Error bars reflect s.d. between animals for one experiment. Similar results were obtained for 4 independent experiments. (D) GLP-1 and PUF-5 expression was assessed by western blot in the indicated strains. Total protein was extracted from the same number of synchronized adult hermaphrodites. puf-5 RNAi strongly reduced both of two PUF-5 bands (arrowheads). GLP-1 runs as multiple diffuse bands (arrowheads) due to glycosylation (Crittenden et al., 1994). Ct: loading control from same blot. (E) Semi quantitative RT-PCR of endogenous glp-1 mRNA extracted from equal numbers of whole worms showed no change in glp-1 mRNA levels in puf-5(RNAi);mpk-1(ga111) animals. Primers distinguished spliced (shown) from unspliced mRNA and DNA products, which were not detected. actin mRNA levels were similar in both samples (data not shown).