RESUMO
The binding of proteins from rabbit reticulocyte lysate to in-vitro-generated beta-globin mRNA and its defined segments was investigated using ultraviolet-cross-linking experiments as well as gel-retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full-length beta-globin mRNA at different positions. The 72-kDa protein is most likely the poly(A)-binding protein and binds, as expected, to the poly(A) tail, whereas the 50-kDa protein exhibits affinity for the trailer region of beta-globin mRNA. The binding region of the 60-kDa protein is located at the 5' end of beta-globin mRNA. The interaction of this protein is dependent on the presence of the 5' cap structure, as indicated by competition experiments using an uncapped beta-globin-mRNA leader segment. Further competition experiments with beta-globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60-kDa protein and the beta-globin mRNA leader.
Assuntos
Proteínas Sanguíneas/metabolismo , Globinas/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/sangue , Reticulócitos/química , Animais , Sequência de Bases , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Peso Molecular , Coelhos , Raios UltravioletaRESUMO
Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.