Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translatio n and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features o f the global secondary structure of a synthetic mRNA and the same intracell ular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enz ymes and chemical reagents to determine single-stranded and hybridized regi ons; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interac tion involving the 5' and 3' untranslated regions and causing a juxtapositi on of the ends of the RNA, was examined further by a useful technique invol ving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was the n done. The modification data support a very similar intracellular structur e. When low reactivity of A and C residues is found in the synthetic RNA, a pproximate to 70% of the same sites are relatively more resistant to DMS mo dification in vivo, A slightly higher sensitivity to DMS is found in vivo f or some of the A and C nucleotides predicted to be hybridized from the synt hetic structural model, With this small mRNA, the translation process and m RNA-binding proteins do not block DMS modifications, and all A and C nucleo tides are modified the same or more strongly than with the synthetic RNA.