Figure 4. Binding of the DAZ-1 protein to fbf mRNAs. (A) Expression pattern of DAZ-1-3xFLAG in the
daz-1; btIs2 strain. Images of an extruded gonad from the
daz-1; btIs2 strain, stained with either anti-FLAG antibody (top) or DAPI (bottom), are shown. Arrows indicate germ nuclei at the diakinesis stage during oogenesis. Arrowhead indicates the distal end of the gonad. Bar, 50 um. (B) Coprecipitation of fbf mRNAs with DAZ-1. (a) Detection of DAZ-1-3xFLAG in the anti-FLAG immunoprecipitate from a btIs2 worm extract by immunoblotting. Tubulin is shown as a negative control. (b) Concentration of
fbf-1 and
fbf-2 mRNAs in the anti-FLAG immunoprecipitate, assayed by RT-PCR. A nonspecific binder
rpl-1 is shown as a control. (C) Features of the 3' UTR sequences of
fbf-1 and
fbf-2 mRNAs. (A/C/U)GUUC sequences are doubly underlined and U-rich tracts, singly. Sequences used as the RNA probes are shown in uppercase. Dotted nucleotides were replaced by the complementary ones in mutant competitors used in E. (D) Binding of DAZ-1 to the 3' UTR of fbf mRNAs. The 3' UTR fragment of
fbf-1,
fbf-2, or
rpl-1 mRNA was biotin labeled, added to a btIs2 worm extract, and pulled down. DAZ-1-FLAG in the biotin-RNA precipitate was detected by immunoblotting using anti-FLAG antibody. S, sense-strand mRNA; AS, antisense-strand RNA. Tubulin is shown as a negative control. (E) In vitro reconstruction of DAZ-1-fbf mRNA complex. Gel retardation assay was performed to monitor the binding of GST-DAZ-1 protein (0-25 ng/ul) to 32P-labeled sense strand 3' UTR RNAs (2 ng/ul). Competitors are unlabeled sense RNA (13-100 ng/ul): F1,
fbf-1; F2,
fbf-2; F1mut,
fbf-1 with GUnC mutated into CAnG; F2mut,
fbf-2 with GUnC mutated into CAnG; and R,
rpl-1.