RESUMO
Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.
Assuntos
Conformação de Ácido Nucleico , Fatores de Iniciação de Peptídeos/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/biossíntese , Saccharomyces cerevisiae/genética , Fator de Iniciação 4E em Eucariotos , Fator de Iniciação Eucariótico 4G , Proteínas Fúngicas/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Substâncias Macromoleculares , Microscopia de Força Atômica , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/ultraestrutura , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA/ultraestrutura , RNA Circular , RNA Fúngico/química , RNA Fúngico/ultraestrutura , RNA Mensageiro/química , RNA Mensageiro/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.
