RESUMO
The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5' untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.
Assuntos
Aminoácidos/metabolismo , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Linhagem Celular , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Camundongos , Conformação de Ácido Nucleico , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/metabolismoRESUMO
In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Biossíntese de Proteínas , Substituição de Aminoácidos , Animais , Sequência de Bases , Sistema Livre de Células , Fator de Iniciação 2 em Eucariotos/genética , Técnicas In Vitro , Camundongos , Fosforilação , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.
