RESUMO
The 5' cap and 3' poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the capâ¢eIF4Eâ¢eIF4Gâ¢PABPâ¢poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the capâ¢eIF4Eâ¢eIF4Gâ¢PABPâ¢poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5' or 3' UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3' UTR, but not the 5' UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3' UTR enhances the cap-poly(A) synergy in translation initiation.
Assuntos
Regiões 3' não Traduzidas , Poli A , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Fator de Iniciação Eucariótico 4G/química , Poli A/química , Proteínas de Ligação a Poli(A)/química , Capuzes de RNA/química , Sistema Livre de Células , Triticum , Saccharomyces cerevisiae , Conformação de Ácido Nucleico , Estabilidade de RNARESUMO
The 5' and 3' termini of RNA play important roles in many cellular processes. Using Förster resonance energy transfer (FRET), we show that mRNAs and lncRNAs have an intrinsic propensity to fold in the absence of proteins into structures in which the 5' end and 3' end are ≤7 nm apart irrespective of mRNA length. Computational estimates suggest that the inherent proximity of the ends is a universal property of most mRNA and lncRNA sequences. Only guanosine-depleted RNA sequences with low sequence complexity are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. While the biological implications remain to be explored, short end-to-end distances could facilitate the binding of protein factors that regulate translation initiation by bridging mRNA 5' and 3' ends. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in RNA in research and biotechnology.
Assuntos
Conformação de Ácido Nucleico , RNA Longo não Codificante/química , RNA Mensageiro/química , Algoritmos , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Humanos , RNA Longo não Codificante/genética , RNA Mensageiro/genéticaRESUMO
Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges.
