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Fig. 6. NO145 mRNA 3' UTRs affect the translation of reporter mRNAs as well as the stability of the encoded proteins in Xenopus oocytes and upon maturation. (A) A firefly luciferase reporter construct (Luc) was either fused to the short (Luc-s) or the long (Luc-l) version of the NO145-3' UTR. The mRNAs were synthesized in vitro with a poly(A)50 tail, capped and subsequently injected at a sixfold excess over Renilla luciferase mRNA in stage VI oocytes. After 4 hours of incubation, the oocytes were homogenized in pools of four oocytes each and the firefly and Renilla luciferase activities were measured. (B) Histogram showing the normalized luciferase activity of two independent experiments each. The translational efficiencies of the three constructs are compared. (C) Capped mRNAs were transcribed in vitro from constructs consisting of a His-tagged version of the NO38 coding sequence followed by either the authentic 3' UTR region of NO38 (His-NO38wt), the short NO145-3' UTR (His-NO38-s) or the long NO145 3' UTR (His-NO38-l). (D) Stage VI oocytes were injected with capped mRNAs synthesized from the different His-NO38 constructs. After incubation overnight, oocyte extracts were analyzed by immunoblotting using the His antibody to allow the specific detection of the reporter proteins encoded by the different mRNA variants. (E) Some of the injected oocytes were induced to maturation by progesterone treatment. Upon GVBD, egg extracts were separated by SDS-PAGE and the corresponding immunoblot was incubated with the His antibody.
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