Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.

Abstract

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpbl-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alters mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MATal, were unstable (t112 < 7 min) and 4, including those encoded by ACT) and PGKI, were stable (t112 > 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.Differences in the decay rates of individual mRNAs can have profound effects on the overall levels of expression of specific genes (80,93). Although the potential importance of mRNA stability as a mechanism for regulating gene expression has been recognized (7, 86), the structures and mechanisms involved in the determination of individual mRNA decay rates have yet to be elucidated. As an approach to understanding the determinants of mRNA stability, we have begun to compare the properties of mRNAs in Dictyostelium discoideum which differ significantly in their respective decay rates (94). In this report, we describe our initial efforts to perform a similar analysis of mRNAs in the yeast Saccharomyces cerevisiae. Our objective was the identification of both stable and unstable yeast mRNAs that were encoded by genes which had already been well characterized. Success in such an endeavor would make it possible to explore the structural determinants of mRNA stability, for example, by analyzing the decay rates of mRNAs transcribed from chimeric genes (25,42,43,81,96,97).Decay rates for both the poly(A)+ RNA population and for individual yeast mRNAs have been measured previously by several different functional or chemical assays. Half-lives ranging from 16 to 23 min have been measured for the average turnover rate of the poly(A)+ RNA population, whereas half-lives of individual mRNAs span a broader range from 1 to over 100 min (3,18,19,35,36,46,47,50,52,54,...

A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.

“…The AMTal mRNA decays rapidly with a tj/2 of 3.5 min ( Fig. 2A), slightly faster than previously described (5 min [18,27]). This small difference presumably reflects the more complete transcriptional shutoff achieved through simultaneous thermal and glucose repression as opposed to simply the thermal repression used previously.…”

“…3A (27). Second, treatment of cells with cycloheximide greatly stabilizes the ALToId mRNA (18). The observation that rare codons are a component of the MIE suggests a means through which translation and decay of the AL4ToLl mRNA may be coupled.…”

“…For each Northern (RNA) gel, 8 ,ug of total RNA was loaded into each lane. mRNA levels were determined by Northern blot analysis (37), using 32p_ labeled random-primed DNA probes (12) and directly counting j decays occurring in each band with a Betascope (18). justed for by stripping and reprobing blots with an oligonucleotide complementary to the 7S small cytoplasmic mRNA, a polymerase III transcript not subject to glucose or the temperature-sensitive polymerase II repression.…”

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

“…Northern blots were quantitated by using a Betagen Blot Analyzer (Betagen, Waltham, MA. ; Herrick et al 1990). Data are expressed as the loglo of the percentage of each RNA remaining versus time at 36~ Reproducibility of mRNA decay rate measurements was ---15%.…”

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the YeastSaccharomyces cerevisiae

“…mRNA decay rates were determined by thermal inactivation of rpb1-1 with the use of a protocol modified from Herrick et al (1990). Cultures of yRP384 carrying either pCD61 or pCD62 were grown in 200 ml of synthetic selective media with 2% glucose at 24°C from an OD 600 of 0.1 to an OD 600 of 0.3-0.4.…”