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Fig. 3
miR-430 represses its target mRNA in the absence of PABP during zebrafish embryogenesis. (A) In situ hybridization detecting pabpc1a mRNA in a 24 hpf zebrafish embryo (purple). (B) Western blot detecting PABP protein in 24 hpf embryos injected with control MO or pabpc1a MO. The membrane was probed with anti-eIF5 antibody as a control. (C) The polysome profiles of control MO-injected (Upper) and pabpc1 MO-injected (Lower) zebrafish embryos at 24 hpf. (D) Analysis of miRNA-mediated target mRNA repression in the presence or absence of PABP. Bright field view (Upper panels), GFP fluorescence (Middle panels; green) and GFP mRNA (Bottom panels; purple) of 24 hpf zebrafish embryos expressing the GFP transgene with three copies of the imperfect target site for the ubiquitously expressed miRNA, miR-430 (miR-430 TS) or with mutated target sites (mut TS). Control MO (left columns) or pabpc1a MO (right columns) was injected as indicated. (E) Quantification of GFP expression levels in 5D. GFP intensity of the embryos with the mut TS and the control MO was set to one. Error bars show SD. Asterisks indicate p < 0.01 compared to the experiment with the mut TS and control MO. (F) The poly(A) tail analysis of the GFP mut TS mRNA with the control MO or pabpc1a MO. The lane +dT shows a completely deadenylated fragment (A0).