Abstract
Glucose repression in Saccharomyces cerevisiae can now be seen to operate at two levels: regulation of transcription of certain genes and control of the half-life of the corresponding mRNAs (Scheffler, I. E., de la Cruz, B. J., and Prieto, S. (1998) Int. J. Biochem Cell Biol. 30, 1175-1193). For example, the steady state levels of SDH2 mRNA and SUC2 mRNA are significantly determined by their differential rates of turnover. A current model for the mechanism of mRNA turnover includes three distinct steps: a rate-limiting deadenylation, removal of the 5'-7-methyl-G (decapping), and 5'-3' exonuclease digestion. We have investigated the same three reactions during glucose-induced degradation of these transcripts. Our results indicate that while decapping (by Dcp1p) and 5'-3' exonuclease digestion (by Xrn1p) are obligatory steps for the rapid degradation of these mRNAs, the dependence on deadenylation is more complicated. At steady state in glycerol these transcripts have very short poly(A) tails but are nevertheless very stable; the addition of glucose causes immediate decapping and degradation without further deadenylation; in contrast, newly made SUC2 mRNA (after a shift from glucose to glycerol) has significantly longer poly(A) tails, and such transcripts are not rapidly degraded upon addition of glucose. A constitutive deadenylation reaction that is independent of the carbon source eventually makes the stability of these transcripts very sensitive to glucose. These results are interpreted in terms of a working hypothesis proposing a competition between translational initiation and decapping influenced by the carbon source. The presence of a long poly(A) tail may also affect this competition in favor of translational initiation and mRNA stabilization.
Highlights
Our laboratory has investigated how glucose regulates the expression of the SDH2 gene encoding the iron-protein subunit of succinate:quinone oxidoreductase of the mitochondrial electron transport chain in Saccharomyces cerevisiae [7,8,9]
When the gene (DCP1) for the decapping enzyme was identified, we examined the kinetics of glucose-induced turnover of SDH2 mRNA in a knock-out compared with its wild type (Fig. 1B)
The rate of SDH2 mRNA turnover in both mutants is strongly affected, especially in the dcp1 strain, where the SDH2 mRNA levels persist for more than 15 min after addition of glucose to the YPG medium. It is the major 5Ј-3Ј exonuclease, Xrn1p accounts only for approximately 30 – 40% of the total RNase activity of crude yeast extracts [30, 31]; it is not surprising that we can still observe some degradation of the SDH2 mRNA in the xrn1 mutant
Summary
Our laboratory has investigated how glucose regulates the expression of the SDH2 gene encoding the iron-protein subunit of succinate:quinone oxidoreductase (complex II) of the mitochondrial electron transport chain in Saccharomyces cerevisiae [7,8,9]. A favored current model has the following three steps: 1) shortening of the poly(A) tail, 2) decapping, and 3) 5Ј-3Ј exonucleotlytic degradation. Poly(A) tail shortening is thought to be a key rate-limiting and obligate step in the decay of constitutively short-lived mRNAs. Factors including Pab1p, the poly(A)-binding protein, and a nuclease associated with Pab1p as well as cis-elements within the mRNA (primarily at the 3Ј end) are thought to regulate the rate of deadenylation. The required role of Dcp1p is documented in the present studies, and it seems clear that the 5Ј cap protects these mRNAs from the exonuclease. It was unresolved whether the glucose signal was targeted at the potentially rate-limiting deadenylation reaction. Does glucose trigger a rapid shortening of the poly(A) tails, resulting in rapid degradation?
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