RESUMO
Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.
Assuntos
Glicoproteínas/genética , Oogênese/genética , Oogênese/fisiologia , Proteínas de Ligação a RNA/metabolismo , Fator de Crescimento Transformador beta/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Superfície/metabolismo , Sequência de Bases , DNA/genética , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Proteína Semelhante a ELAV 2 , Feminino , Técnicas In Vitro , Oócitos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
We have shown that ElrA and ElrB, Xenopus ELAV homologues, bind the Vg1 mRNA 3'UTR translation element in Xenopus oocytes and implicated ElrB in mediating translational repression during oogenesis. Here we report that, while ElrA and ElrB are 69% identical and both exhibit RNA binding in the nM range, recombinant ElrB, but not ElrA, is able to oligomerise. This oligomerisation is also seen with the endogenous protein. Both RNA binding and oligomerisation require the linker region flanked with two RNA recognition motifs. Our data demonstrate a novel and unique property of ElrB which may be important for its function as a translational regulator.
Assuntos
Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Proteína Semelhante a ELAV 2 , Oócitos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
RNA-binding proteins play a major part in the control of gene expression during early development. At this stage, the majority of regulation occurs at the levels of translation and RNA localization. These processes are, in general, mediated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated regions of their target RNAs. Although initial work concentrated on the analysis of these sequences and their trans-acting factors, we are now beginning to gain an understanding of the mechanisms by which some of these proteins function. In this review, we will describe a number of different families of RNA-binding proteins, grouping them together on the basis of common regulatory strategies, and emphasizing the recurrent themes that occur, both across different species and as a response to different biological problems.