Rapid mRNA degradation in yeast can proceed independently of translational elongation.

We have exploited a modular cat reporter system (Vega Laso, M. R., Zhu, D., Sagliocco, F. A., Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462) to investigate the relationship between mRNA structure, translation, and stability in the yeast Saccharomyces cerevisiae. The stability of the cat mRNA was not influenced by changes in the length and nucleotide sequence of the 5'-leader, but was affected by the formation of stable 5'-secondary structures (> -15 kcal.mol-1). Cat mRNA stability changed only slightly when the CYC1 3'-trailer was replaced with PGK1 sequences, and was influenced by some secondary structures in the 3'-trailer. Secondary structures formed by interactions between the 5'-leader and 3'-trailer increased the stability of the cat mRNA. However, all of the cat mRNAs studied were intrinsically unstable, having half-lives between 4 and 14 min. The translatability of the cat mRNAs did not correlate with their half-life, and their decay was not blocked by cycloheximide. Therefore, the rapid degradation of the cat mRNA does not seem to depend on translational elongation and is not related in any obvious way to the rate of translational initiation. Furthermore, sequences in the 3'-trailer do not program the rapid decay of the cat mRNA. We discuss the implications of these data in the light of current models of mRNA degradation pathways.

The influence of 5'-secondary structures upon ribosome binding to mRNA during translation in yeast.

The influence of 5'-secondary structure formation and 5'-leader length upon mRNA translation in yeast has been analyzed using a closely related set of cat mRNAs (Vega Laso, M. R., Zhu, D., Sagliocco, F. A., Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462). A cat mRNA with a relatively short unstructured 5'-leader (22 bases) had a ribosome loading about half that of a cat mRNA with an unstructured 5'-leader of 77 bases. The introduction of 5'-secondary structures at various positions throughout the 5'-leader of the cat mRNA inhibited translation initiation, the degree of inhibition being largely dependent upon the thermodynamic stability of the structure. Each mRNA carrying a 5'-secondary structure had a biphasic polysome distribution, indicating that the mRNA molecules were distributed between untranslated and well translated subpopulations. This suggests that once 5'-secondary structures are unwound, they reform slowly relative to the rate of translation initiation in yeast. Untranslated mRNA accumulated in 43 S preinitiation complexes, even when there were only 5 bases between the 5'-cap and the base of the hairpin. The data are consistent with the scanning hypothesis (Kozak, M. (1989) J. Cell. Biol. 108, 229-241) and suggest that 40 S ribosomal subunits bind to mRNA early in the scanning process, probably before mRNA unwinding has taken place.

Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader.

A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae. We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady-state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9. mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure. A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98%. A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66%. The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader. An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined. Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo. The stem-loop structures were far less inhibitory in a reticulocyte lysate system. Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression.