RESUMEN
Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biosíntesis de Proteínas/genética , Multimerización de Proteína/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Chlorocebus aethiops , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Mutación Puntual/genética , Proteínas de Unión a Poli(A)/genética , Unión Proteica/genética , ARN Mensajero/genética , ARN Viral/genética , Rotavirus/genética , Infecciones por Rotavirus/virología , Alineación de Secuencia , Replicación Viral/genéticaRESUMEN
Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5' end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5' rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5' end and a poly(A) tail at the 3' end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes.
Asunto(s)
Leishmania major/genética , Proteínas Protozoarias/metabolismo , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , Aminoacil-ARN de Transferencia/genética , Leishmania major/enzimología , Leishmania major/metabolismo , Poliadenilación , Empalme del ARNRESUMEN
OBJECTIVES: The alliance theory holds that homosexual behavior in humans may have evolved because it reinforced same-sex alliances which contributed to survival and reproduction. The present study was designed to test this evolutionary hypothesis in view of the strongly negative perception of homosexual behavior. It was predicted that targets who engaged in homosexual behavior would be perceived as likely to achieve greater social status and reproductive opportunities when the behavior reinforced an alliance that led to increased social opportunities. METHODS: Three hundred sixteen men and women read scenarios in which a target engaged in homosexual behavior and then answered questions about the target's future social status and reproductive opportunities as measured by number of sexual partners. The data were analyzed in two 3 (social outcome: positive vs. neutral vs. negative) x 2 (gender of target) x 2 (gender of participant) analyses of variance. RESULTS: The results supported the predictions and demonstrated that targets were perceived as likely to have the greatest social status and reproductive opportunities in the positive outcome condition. CONCLUSION: The alliance theory of homosexual behavior may have heuristic value for the evolutionary study of homosexual behavior and its perception.