The nuclear experience of CPE does not mediate mRNA nuclear export or cytoplasmic deadenylation. (A) Diagram of experiment procedure for comparison of RNA export between CPE-containing and CPE-lacking luciferase mRNA. (B) Cytoplasmic luciferase mRNA levels following plasmid injection as determined by radioactive semiquantitative RT-PCR (upper panel). The relative mRNA levels are graphed in the lower panel. (C) Diagram of experiment procedure for comparison of deadenylation between cytoplasm-injected and nucleus-injected cyclin B1 mRNA. (D) Deadenylation assay. A radiolabled and polyadenylated partial cyclin B1 mRNA was injected into the nucleus or cytoplasm of oocytes; after overnight incubation, the cytoplasmic fraction was collected for RNA extraction and analysis on a denaturing polyacrylamide gel. (E) Deadenylation assay of CPE-containing RNA. Oocytes were injected with in vitro transcribed RNA or plasmid DNA; RNA collected over several hours was analyzed by ligation-mediated PAT assay (see Materials and Methods). Lower panels are ethidium bromide stained agarose gels showing RT-PCR products of cyclin B1 RNA; cyclin B1 mRNA started to accumulate ∼3 h after injection in the nucleus and ∼6 h in the cytoplasm. Note that because the RT-PCR does not distinguish endogenous from ectopic cyclin B1 3′ UTR, a band is present in the cytoplasmic fraction of noninjected oocytes.
