Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2780-2784, April 1990 Biochemistry Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MATal gene are involved in promoting rapid mRNA decay in yeast (cis-acting instability element/rare codons/ribosome) RoY PARKERt AND ALLAN JACOBSON Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655 Communicated by Gerald R. Fink, January 16, 1990 (received for review November 20, 1989) ABSTRACT In yeast, the mRNA encoded by the MATal vation consistent with the rapid transition to a new set of gene is unstable (tl2 = 5 min) and the mRNAs encoded by the specific functions that occurs during mating-type switching ACTI gene (til2 = 30 min) and the PGKI gene (t.l2 = 45 min) are (18). To understand the structural basis for the instability of stable. To understand the RNA structural features that dictate the MATal mRNA, we have constructed several chimeras mRNA decay rates in yeast, we have constructed PGKI/ between the MATal gene and the PGKI and ACT] genes, MATal and ACTJ/MATal gene fusions and analyzed the both of which encode stable mRNAs. The decay rates of the decay rates of the resultant chimeric transcripts. Fusion of a hybrid mRNAs identify a 42-nucleotide (nt) coding-region MATal segment containing 73% of the coding region and the segment that is required for rapid decay. The insertion of a 3' untranslated region to either of the stable genes is sufficient nonsense codon immediately 5' to this 42-nt segment pre- to cause rapid decay ofthe chimeric mRNAs (t.12 = 6-7.5 min). vents rapid mRNA turnover, suggesting that the decay ofthe Sequences required for this rapid decay are not found in the MATal mRNA is promoted by the translocation of ribo- MATal 3' untranslated region but are located within a 42- somes through a specific region of the coding sequence. nucleotide segment of the coding region that has a high content (8 out of 14) ofrare codons. Introduction ofa translational stop codon upstream of this region stabilizes the hybrid mRNAs, MATERIALS AND METHODS indicating that the rapid decay promoted by these sequences is Strain and Media. The yeast strain was Y262 (MATa, his4- dependent on ribosomal translocation. 519, ura3-52, rpbl-l; provided by M. Nonet and R. Young; ref. 19). Synthetic media lacking uracil (20) were used to select for Individual mRNAs are degraded at rates that vary by more and maintain plasmids introduced by transformation (21). than an order of magnitude. Although such differences can Plasmid Constructions. The DNA sequences of all junc- affect the intrinsic level ofgene expression and serve as a site tions within the translated regions of gene fusions were of posttranscriptional regulation, little is known about the determined by the dideoxy method (22), using oligonucleo- features of mRNAs that dictate their respective decay rates. tides complementary to flanking sequences as primers. Plas- Initially, it was suggested that the stability of mRNAs is related to basic mRNA properties such as size (1, 2), poly(A) mid constructions (23) were as follows. tail length (3), and translational efficiency (4, 5). However, Pal. Plasmid pRIP4 (a pUC19 derivative containing the several comparisons have failed to reveal any correlation PGKI gene) was cut with Bgl II. Overhanging ends were between these variables and the rate of mRNA turnover, filled in with Klenow enzyme and the DNA was recut with suggesting that general mRNA features are not the primary HindIII. The 1.6-kilobase (kb) EcoRV-HindIII fragment determinants of mRNA decay rate (6, 7). An alternative from pl.9, a subclone of MATal in pBR322 (ref. 25; obtained explanation for differences in decay rate is that unstable from K. Tatchell), was inserted into these sites to yield mRNAs contain specific sequences that promote their rec- pUC19.Pal. The resulting hybrid gene was then subcloned ognition by the cellular turnover machinery. This view is into a yeast centromere vector, pRIPlH [a derivative of supported by recent experiments in which specific sequences pUN50 (26) in which the HindIII site flanking the URA3 gene derived from unstable metazoan mRNAs promoted rapid had been filled in] to yield pRIPlH.Pal. decay when transferred to stable mRNAs. To date, these Aal. First, the Cla I-HindIlI fragment of the PGKI gene "instability elements" have been found in the 3' untranslated in pRIP4 was replaced with the Asu II-HindIII fragment of region (UTR) (8-11) and in coding regions (12-15). Although the MATal gene to yield pUC19.Pal*. The BamHI-HindIII the mechanisms by which such elements promote mRNA fragment of this hybrid gene was then subcloned into the turnover are unknown, there appears to be a close link BamHI and HindIII sites of pRIP1H to yield pRIPlH.Pal*. between the translation process and mRNA decay (16, 17). This plasmid was then digested with BamHI and partially In this paper we describe experiments to identify specific digested with EcoRV (to liberate the remaining PGKI frag- sequences that promote mRNA decay in the yeast Saccha- ment and a small segment of the MATal coding region) and romyces cerevisiae. We recently reported the decay rates of the BamHI-HindIII fragment of the actin gene from pYactl a number of yeast mRNAs, including both stable and unsta- (27) was inserted. The resulting plasmid was designated ble species (18). In agreement with previous work, no cor- pRIPlH.Aal. relation was found between mRNA decay rate and several P(ut)al. pRIP4 (see above, Pal) was cut with Cla I, filled general mRNA properties. Among the unstable mRNAs in with Klenow enzyme, digested with HindIII, and ligated to identified were those encoded by genes involved in mating the 1.1-kb Sca I-HindIII fragment of MATal isolated from (e.g., MATa), STE2, STE3, MFal, and MFA2), an obser- pl.9. This plasmid was designated pRIPl.P(ut)al. The publication costs of this article were defrayed in part by page charge Abbreviations: nt, nucleotide(s); UTR, untranslated region. payment. This article must therefore be hereby marked "'advertisement" tCurrent address: University Department of Molecular and Cellular in accordance with 18 U.S.C. §1734 solely to indicate this fact. Biology, University of Arizona, Tuscon, AZ 85721. 2780 Downloaded by guest on September 27, 2021 Biochemistry: Parker and Jacobson Proc. NatL. Acad. Sci. USA 87 (1990) 2781 PalP. The 0.26-kb Cla I (filled-in)-HindIII fragment of Minutes at 36-C PGKI that contains the 3' UTR was inserted into pRIPlH.- Pal that had been digested partially with Sca I (site just 0 5 10 20 30 40 50 60 downstream ofthe MATal translational stop) and completely with HindIII, yielding pRIPlH.PalP. ;ii.- so Deletions of Pal. pUC19.Pal.UAG (see below) was cut PGK1 --..w 45 min with Bgl II and then treated with BAL-31 nuclease for various MA&-.-a 5 min times. Single-stranded overhangs were filled in with Klenow MATa 1 _ enzyme and then ligated to Bgl II linkers (no. 1001; New England Biolabs). The DNA was then cut with HindIII and the fragments containing the MATal region were purified and 100 0 inserted into Bgl II/HindIII-cut pRIPlH.Pal.UAG. Plas- mids these deletions are as U O containing designated pRIPlH.- CZ PalAx, where x is the number of nucleotides deleted in the 3' E direction from the EcoRV site of the MATal gene. For a) the frame was 10 pRIPlH.PalA52, reading restored, following z sequencing, by filling in the Bgl II site at the new junction CC between the PGKI and MATal sequences. oPso Pal. UAG. pUC19.Pal* (see above) was cut with EcoRV and ligated to the linker 5'-CTAGATCTAG-3'. The DNA was 1 then cut with Bgl II (cutting the linker and the Bgl II site in 20 40 60 the PGKI sequences) and the plasmid was ligated to itself. Minutes at 36°C This plasmid, pUC19.Pal.UAG, was cut with BamHI and HindIII, and the fragment containing the hybrid gene was FIG. 1. Decay of stable and unstable mRNAs in yeast. Relative inserted into the BamHI and HindIII sites of levels of the PGKJ and MATal mRNAs were measured at different pRIPlHB (a times after a shift to 36°( in a temperature-sensitive RNA polymer- derivative ofpRIPlH in which the Bgl II site in the backbone ase II mutant. (Upper) Northern analysis ofmRNA levels at different had been filled in with Klenow enzyme), yielding the plasmid times after the temperature shift. The probe for the PGKJ mRNA pRIPlHB6.Pal.UAG. was a random-primed Dra 1-HindlIl restriction fragment containing Aal. UAG. The 1.4-kb BamHI-HindIII (filled-in) fragment the entire PGKI coding sequence. The probe for the MATal mRNA containing the 5' portion of the ACT) gene was inserted into was the same as for Fig. 2. (Lower) Quantitation ofthe Northern blot. the BamHI and Bgl II (overhang removed with mung bean mRNA levels are normalized to the level at time zero. *, MATal; o, nuclease) sites of pRIPlHB.Pal.UAG to yield pRIPlHB6.- PGKI. Half-lives (45 min and 5 min) calculated from the slopes are Aal.UAG. shown to the right of the autoradiogram. mRNA Decay Measurements, RNA Preparation, and RNA Analysis. mRNA decay rates were measured as described (18). mRNA (see schematic, Fig. 2). The only MATal sequences In brief, transcription was inhibited by thermal inactivation of absent from this hybrid are the 21-nt 5' UTR and the initial RNA polymerase II and mRNA levels were quantitated by 140 nt of coding sequence. Northern blotting (28) with DNA probes labeled to high Decay of Pal mRNA was assayed by transferring the specific activity (29).