The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer.
Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2-Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2-Xp54 tethered to the reporter mRNA 3'-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3'UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Nicola
N
Minshall
Nancy
N
Standart
D000818:Animals; D053487:DEAD-box RNA Helicases; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D008540:Meiosis; D008957:Models, Genetic; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010063:Ovum / Q000378:metabolism; D014176:Protein Biosynthesis; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014981:Xenopus / Q000235:genetics* / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D039221:mRNA Cleavage and Polyadenylation Factors
C490388:Cpeb1 protein, Xenopus; D039561:Eukaryotic Initiation Factor-4E; D046911:Macromolecular Substances; D012333:RNA, Messenger; D014157:Transcription Factors; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C486437:Xp54 RNA helicase, Xenopus; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases