RESUMEN
Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.
Asunto(s)
Complejos Multiproteicos/metabolismo , Proteínas de Unión al ARN/metabolismo , Selenocisteína/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Codón de Terminación , Citoplasma/metabolismo , Humanos , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Serina/genética , ARN de Transferencia de Serina/metabolismo , Proteínas de Unión al ARN/genética , Selenoproteínas/biosíntesis , Selenoproteínas/metabolismoAsunto(s)
Elementos Transponibles de ADN , Proteínas/genética , Selenocisteína/genética , Animales , Western Blotting , Línea Celular , Codón/genética , Codón de Terminación/genética , Bases de Datos de Ácidos Nucleicos , Expresión Génica , Humanos , Yoduro Peroxidasa/genética , Biosíntesis de Proteínas , Radioisótopos de Selenio , Selenocisteína/metabolismo , Selenoproteínas , TransfecciónRESUMEN
SECIS elements recode UGA codons from "stop" to "sense." These RNA secondary structures, present in eukaryotic selenoprotein mRNA 3' untranslated regions, recruit a SECIS binding protein, which recruits a selenocysteine-specific elongation factor-tRNA complex. Elucidation of the assembly of this multicomponent complex is crucial to understanding the mechanism of selenocysteine incorporation. Coprecipitation studies identified the C-terminal 64 amino acids of the elongation factor as sufficient for interaction with the SECIS binding protein. Selenocysteyl-tRNA is required for this interaction; the two factors do not coprecipitate in its absence. Finally, through promoting this interaction, selenocysteyl-tRNA stabilizes the C-terminal domain of the elongation factor. We suggest that the coupling effect is critical to preventing nonproductive decoding attempts and hence forms a basis for effective selenoprotein synthesis.