Oocyte selection of mutations affecting cytoplasmic polyadenylation of maternal mRNAs.
Translational activation by cytoplasmic polyadenylation is a conserved mechanism in metazoan early development. In Xenopus and mouse, the regulatory sequences that control this process during oocyte meiotic maturation have been identified in the 3' untranslated region (3'-UTR) of a class of maternal messenger RNAs (mRNAs). In this report, we have investigated sequences controlling cytoplasmic polyadenylation of a mouse maternal mRNA. Pools of RNAs, transcribed from DNA randomly mutated by a PCR-based method, were micro-injected into the cytoplasm of mouse primary oocytes to allow in vivo selection of inefficiently polyadenylated transcripts. After oocyte maturation, the nonelongated RNAs were gel-isolated, and single base substitutions that alter poly(A) addition were identified. Analysis of these mutant RNAs identified single nucleotides that influence efficiency of cytoplasmic polyadenylation during mouse oocyte maturation. In addition, this strategy should facilitate identification of yet unknown sequence elements responsible for basic biological mechanisms during and after early development.
Verrotti
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AC
Department of Pharmacology, University Medical Center at Stony Brook, NY 11794-8651, USA.
Strickland
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S
D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D008540:Meiosis / Q000235:genetics; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D017354:Point Mutation; D011061:Poly A / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011061:Poly A; D010959:Tissue Plasminogen Activator