ABSTRACT
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.
Subject(s)
Nucleic Acid Conformation , Peptide Initiation Factors/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , RNA/biosynthesis , Saccharomyces cerevisiae/genetics , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factor-4G , Fungal Proteins/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Macromolecular Substances , Microscopy, Atomic Force , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Initiation Factors/genetics , Peptide Initiation Factors/ultrastructure , Poly(A)-Binding Proteins , Protein Biosynthesis , RNA/ultrastructure , RNA, Circular , RNA, Fungal/chemistry , RNA, Fungal/ultrastructure , RNA, Messenger/chemistry , RNA, Messenger/ultrastructure , RNA-Binding Proteins/ultrastructure , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae ProteinsABSTRACT
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.
