A 250-nucleotide UA-rich element in the 3′ untranslated region of Xenopus laevis Vg1 mRNA represses translation both in vivo and in vitro

Abstract

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. Vg1 protein does not appear until late stage IV, after localization is complete. To determine whether Vg1 translation is regulated by cytoplasmic polyadenylation, the RACE-PAT method was used. Vg1 mRNA has a constant poly(A) tail throughout oogenesis, precluding a role for cytoplasmic polyadenylation. To identify cis-acting elements involved in Vg1 translational control, the Vg1 3′ UTR was inserted downstream of the luciferase ORF and in vitro transcribed, adenylated mRNA injected into stage III or stage VI oocytes. The Vg1 3′ UTR repressed luciferase translation in both stages. Deletion analysis of the Vg1 3′ UTR revealed that a 250-nt UA-rich fragment, the Vg1 translational element or VTE, which lies 118 nt downstream of the Vg1 localization element, could repress translation as well as the full-length Vg1 3′ UTR. Poly(A)-dependent translation is not necessary for repression as nonadenylated mRNAs are also repressed, but cap-dependent translation is required as introduction of the classical swine fever virus IRES upstream of the luciferase coding region prevents repression by the VTE. Repression by the Vg1 3′ UTR has been reproduced in Xenopus oocyte in vitro translation extracts, which show a 10–25-fold synergy between the cap and poly(A) tail. A number of proteins UV crosslink to the VTE including FRGY2 and proteins of 36, 42, 45, and 60 kDa. The abundance of p42, p45, and p60 is strikingly higher in stages I–III than in later stages, consistent with a possible role for these proteins in Vg1 translational control.