MS2-xPat1a and -xPat1b repress translation when tethered and interact with CPEB RNP components. (A) xPat1 proteins repress translation when tethered. mRNAs encoding MS2-tagged xPat1a, -xPat1b, and control mRNAs encoding MS2, ePAB, and 4E-T were injected into stage VI oocytes. Six hours after the first injection, firefly luciferase mRNA reporter (Fluc) (m7GpppG-capped but nonpolyadenylated) containing 3′ UTR MS2 hairpins, was coinjected with Renilla luciferase (Rluc), used as an internal control (dark gray). Firely luciferase reporter mRNA lacking the MS2 hairpins was used as a control (light gray). The ratios of the luciferase activities were normalized to the one observed with MS2 alone. Three independent experiments were performed and standard deviation bars are shown. (B) qPCR showing equal levels of the injected RNAs. The qPCR was first normalized to GAPDH levels, and the relative ratio between luciferase and Renilla reporter mRNAs was subsequently normalized to MS2. Three independent experiments were performed and standard error bars are shown. (C) CPEB antibody immunoprecipitates tagged xPat1a and xPat1b. mRNAs encoding the MS2-Flag–tagged xPat1a, xPat1b were injected in stage VI oocytes. The immunoprecipitated proteins were analyzed by Western blotting with the indicated antibodies. (D) mRNAs encoding the MS2-Flag–tagged xPat1a, xPat1b were injected in stage VI oocytes, some of which were matured with progesterone into eggs. Immunoprecipitation was carried out using Flag antibody, and the Western blot was analyzed with the indicated antibodies. *Phosphorylated form of Flag-xPat1b. One stage VI oocyte was loaded as an input (2%).
