Molec. gen. Genet. 170, 137-144 (1979) © by Springer-Verlag 1979
The Half-Life of mRNA in Saccharomyces cerevisiae Li-Li Chia and Calvin McLaughlin* Department of BiologicalChemistry,Universityof California,Irvine, Irvine, California92717, USA
Summary. The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37° C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts 136 cell culture and pulse-labelling with [~5S]methionine at various time intervals after the shift to 37° C from 23 ° C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for their incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic decay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.
Introduction
The half-life of yeast mRNA has been determined by several investigators. Hartwell et al. (1970) measured the entrance kinetics of newly synthesized RNA into polyribosomes in the presence of cycloheximide and Hutchison et al. (1969) measured the kinetics of the decay of polyribosomes in mutant ts136. These experiments suggested a value of 20 to 23 min for the half-life of mRNA in yeast. Tonnesen and Friesen (1973) studied the rate of loss of the ability to incorporate [3H]leucine into proteins in the presence of either daunomycin or ethidium bromide and determined the average half-life of total mRNA to be about 22 min. Hynes and Phillips (1976), and Petersen et al. (1976) studied the kinetics of incorporation of labelled ade-
nine into polyadenylate containing RNA in yeast and calculated an average mRNA half-life of 16 to i7 min. Kuo et al. (1973) studied the disappearance of the capacity of yeast to synthesize various enzymes after the cells had been treated with lomofungin, an RNA polymerase inhibitor. They reported a 20 min half-life for the messenger coding for invertase, e-glucosidase and acid phosphatase. A 15 min half-life was reported for homocysteine synthetase mRNA by Surdin-Kerjan and de Robichon-Szulamajster (1975), using a similar approach, On the other hand, Bossinger and Cooper (1977) demonstrated that the synthetic capacity to produce arginase decays with a half-life of 4 to 5 min at 30° C. The results of similar experiments conducted with allophanate hydrolase (Lawther and Cooper, 1975) and ornithine carbamoyltransferase (Messenguy and Cooper, 1977) have yielded half-life values of 3, and 5 to 7 rain, respectively. Gorenstein and Warner (1976) reported that the mRNA coding for several individual ribosomal proteins decayed with a half-life of 9 to 12 min at 36° C in ts136 and in a mutant, ts368, in which no ribosomes are made at the restrictive temperature. Sripati et al. (1976) showed that, in yeast, there are two classes of mRNA molecules with 5' termini of mTGpppA and mTGpppG, respectively, having a ratio of 3 : 1. It has been found that these two classes of mRNA have different stabilities. This discovery was based on measuring the disappearance of aH from 5' terminal caps of mRNA in cells labelled uniformly with [14C]adenine and pulsed with [3H](CH3)methionine. Those mRNAs with mVGpppG decayed more rapidly, yet neither class showed a simple exponential decay and each contained two roughly equal components. These components had half-lives of 3-5% and 15-20% of the doubling time of the cell (Sripati and Warner, 1977). Thus, there is reasonable agreement among a number of laboratories using different methods that the average half-life for bulk
0026-8925/79/0170/0137/$01.60
138
mRNA is around 20 min for S. cerevisiae growing with a generation time of around three hours. However, studies on the half-life for mRNAs of individual proteins have presented a wide range of values, most of which have been significantly lower than the halflife values for total mRNA. To investigate this discrepancy, we have determined the half-life of individual yeast mRNAs. Because cultures of mutant ts136 at a nonpermissive temperature undergo a rapid inhibition of RNA transport and a slow decay in the rate of protein synthesis, we were able to follow the functional half-life of pre-existing mRNA uncomplicated by the entrance of newly synthesized RNA into the cytoplasm (Hutchison etal., 1969). The rate of protein synthesis was measured by the incorporation of [35S] methionine into proteins and uniformly 3Hlabelled cells were added to the pulse-labelled cells to serve as an internal control for recovery at various steps of sample manipulations. Using O'Farrell's twodimensional gel electrophoretic technique (O'Farrell, 1975) to separate the cellular proteins, the ratio of 3sS to 3H of each protein then was taken as the quantitative measurement of the relative rate of protein synthesis. The high resolution of O'Farrell's two-dimensional gel electrophoretic system made possible the direct examination of the decay rate of a large number of mRNAs coding for individual polypeptide chains. Materials and Methods Strains and Media. A haploid strain of Saccharomyces cerevisiae a ade 1 ade z ural tyrl his1 lys2 gall rnal), mutant ts136, was used in all experiments. Cultures were grown on a rotary shaker in media containing per liter: 6.7 g yeast nitrogen base minus amino acids (Difco), 21 mg each of all amino acids with the exception of methionine and cysteine, 100 mg adenine, 100 mg uracil, 20 g glucose, 200 mg each of yeast extract and of peptone, 10 g succihate, and 6 g sodium hydroxide. The doubling time was approximately 200 min. Cells were switched from 23 ° C to 37° C as follows: One half of the midlog phase culture was rapidly filtered through a 5 cm diameter Millipore filter (pore size 0.45 g) and the filtered media (without cells) was transferred to a 37° C water bath where 5 min was allowed for temperature equilibration. The second half of the midlog phase culture was filtered through another clean Millipore filter and then rapidly suspended in the prewarmed media described above. Although this procedure results in only half of the cells being transferred from 23 ° C to 37° C, it has the advantage of transferring cells from one temperature to another in a minimum length of time and into a preconditioned media having exactly the same composition.
Radioactive Labelling. Equal volumes (10 ml) of an exponentially growing ts136 culture were withdrawn at various intervals after the shift to 37° C and pulse-labelled for 5 min with 160 laCi [3~S]methionine (specific activity, 400 Ci/mmole from Amersham). The incorporation was linear over this labelling period. The pulses were terminated by adding cycloheximide (final concentration,
L.-L. Chia and C. McLaughlin: Half-Life of Yeast mRNA 0.5 mg/ml) and ice to the media. 3H-labelled cells were obtained from log phase culture labelled for 20 h with 3H-protein hydrolysate (30 gCi/ml). An equal volume (5 ml) of 3H-amino acid labelled cells was added to each of the pulse samples and used as an internal standard for recovery at various steps of sample manipulation.
Sample Preparation and Electrophoresis. Cells collected by centrifugation and pulse-labelled with [35S]methionine were grouped with cells labelled with 3H-amino acids and then resuspended in a small amount (< 100 gl) of ice cold water in a glass tube. To this was added 0.2 volumes of RNase-DNase solution (20 mM CaC12, 50 mM MgCI2, 0.5 mg/ml RNase, 1 mg/ml DNase, 0.5 M Tris, pH 7.0) and acid-washed glass beads (0.45 mm) level to the meniscus. The cells were lysed by vortexing three times for 30 s each at maximum speed on a vortex mixer. The tubes were cooled on ice between each vortexing. A 0.1 volume of RNase-DNase solution and a 0.1 volume of solution containing 0.1% sodium dodecyl sulfate (SDS) and 1% fi-mercaptoethanol were added. After 15 min, the sample was taken off ice and solid urea was added to a concentration of 9.5 M urea, followed by one volume of sample buffer containing 9.5 M urea, 4% Nonidet P-40 (w/v), 5% ~-mercaptoethanol, 2% ampholines (comprising 0.8% pH range 5 to 7, 0.8% pH range 5 to 8, and 0.4% pH range 3 to 10). The samples were maintained at room temperature and immediately loaded onto the gel. Two-Dimensional Gel Electrophoresis. Electrophoresis was performed according to the procedure of O'Farrell (1975) with the following modifications: The isoelectric focusing gel was 3% acrylamide and the sample overlay contained 9 M urea, 4% Nonidet P-40, 5% fi-mercaptoethanol, and 2% ampholines. The firstdimension gels were focused for 6,000 v-h. A 10% acrylamide gel with SDS was used for the second-dimension, The gels were stained, dried, and exposed to No-screen medical x-ray films according to standard procedures. To measure the radioactivity of a protein spot, the spot was cut from the dried gel and digested with 25 gl H20, 160 gl protosol and 4 ml toluene based scintillation fluid. The mixture was stored at room temperature with occasional shaking for three days before counting in a scintillation counter. The ratio of 3~S/3H counts in individual spots represented the relative rate of synthesis of each polypeptide. Polyribosome Analysis. Ribosomal fractions were prepared from a yeast whole cell culture by vortexing with glass beads followed by zone sedimentation in 10-40% (w/w) linear sucrose gradient in a Spinco model SW 56 ti rotor as previously described (Hartwell and McLaughlin, 1968).
Results Decay of Polyribosomes in ts136 at 37 ° C. The rapid
inhibition of RNA transport into the cytoplasm in mutant ts136 results in the decay of the mRNA in polyribosomes with the consequent conversion of polyribosomes into monoribosomes. Figure 1 shows the polyribosome profiles from whole cell cultures of the mutant incubated at 23° C and 37° C for 1 h, in which 71% and 12.8%, respectively, of the ribosomes were in polyribosomes. The decay of cytoplasmic polyribosomes in ts136 at 37° C is characteristic of the instability of existing mRNA. After the shift
L.-L. Chia and C. McLaughlin: Half-Life of Yeast m R N A
139
I
O
O
~
37 °
23° 3A
I
2 o
,.o (xl
1
," 70
45
m ©
"
.:
"1"
31 -
=~K)
i ". "
1~
7.~
4~
"
-
r~" '%
J
"75
25
77
-~ X ,,,=.4
C)
&
,
~
,~44
-,--
12 Fig. 4. Map of a yeast protein two-dimensional gel. This map was prepared from the autoradiogram of Figure 3A. Numbers were assigned to analyzed proteins. Molecular weight was determined by co-electrophoresis of bovine serum (67,000 daltons), ovalbumin (45,000 daltons), chymotrypsinogen (25,100 daltons), and hemoglobin (17,000 daItons
b .5 .2 t-~
II
-J
The Half-Life of mRNA in Saccharomyces cerevisiae Li-Li Chia and Calvin McLaughlin* Department of BiologicalChemistry,Universityof California,Irvine, Irvine, California92717, USA
Summary. The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37° C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts 136 cell culture and pulse-labelling with [~5S]methionine at various time intervals after the shift to 37° C from 23 ° C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for their incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic decay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.
Introduction
The half-life of yeast mRNA has been determined by several investigators. Hartwell et al. (1970) measured the entrance kinetics of newly synthesized RNA into polyribosomes in the presence of cycloheximide and Hutchison et al. (1969) measured the kinetics of the decay of polyribosomes in mutant ts136. These experiments suggested a value of 20 to 23 min for the half-life of mRNA in yeast. Tonnesen and Friesen (1973) studied the rate of loss of the ability to incorporate [3H]leucine into proteins in the presence of either daunomycin or ethidium bromide and determined the average half-life of total mRNA to be about 22 min. Hynes and Phillips (1976), and Petersen et al. (1976) studied the kinetics of incorporation of labelled ade-
nine into polyadenylate containing RNA in yeast and calculated an average mRNA half-life of 16 to i7 min. Kuo et al. (1973) studied the disappearance of the capacity of yeast to synthesize various enzymes after the cells had been treated with lomofungin, an RNA polymerase inhibitor. They reported a 20 min half-life for the messenger coding for invertase, e-glucosidase and acid phosphatase. A 15 min half-life was reported for homocysteine synthetase mRNA by Surdin-Kerjan and de Robichon-Szulamajster (1975), using a similar approach, On the other hand, Bossinger and Cooper (1977) demonstrated that the synthetic capacity to produce arginase decays with a half-life of 4 to 5 min at 30° C. The results of similar experiments conducted with allophanate hydrolase (Lawther and Cooper, 1975) and ornithine carbamoyltransferase (Messenguy and Cooper, 1977) have yielded half-life values of 3, and 5 to 7 rain, respectively. Gorenstein and Warner (1976) reported that the mRNA coding for several individual ribosomal proteins decayed with a half-life of 9 to 12 min at 36° C in ts136 and in a mutant, ts368, in which no ribosomes are made at the restrictive temperature. Sripati et al. (1976) showed that, in yeast, there are two classes of mRNA molecules with 5' termini of mTGpppA and mTGpppG, respectively, having a ratio of 3 : 1. It has been found that these two classes of mRNA have different stabilities. This discovery was based on measuring the disappearance of aH from 5' terminal caps of mRNA in cells labelled uniformly with [14C]adenine and pulsed with [3H](CH3)methionine. Those mRNAs with mVGpppG decayed more rapidly, yet neither class showed a simple exponential decay and each contained two roughly equal components. These components had half-lives of 3-5% and 15-20% of the doubling time of the cell (Sripati and Warner, 1977). Thus, there is reasonable agreement among a number of laboratories using different methods that the average half-life for bulk
0026-8925/79/0170/0137/$01.60
138
mRNA is around 20 min for S. cerevisiae growing with a generation time of around three hours. However, studies on the half-life for mRNAs of individual proteins have presented a wide range of values, most of which have been significantly lower than the halflife values for total mRNA. To investigate this discrepancy, we have determined the half-life of individual yeast mRNAs. Because cultures of mutant ts136 at a nonpermissive temperature undergo a rapid inhibition of RNA transport and a slow decay in the rate of protein synthesis, we were able to follow the functional half-life of pre-existing mRNA uncomplicated by the entrance of newly synthesized RNA into the cytoplasm (Hutchison etal., 1969). The rate of protein synthesis was measured by the incorporation of [35S] methionine into proteins and uniformly 3Hlabelled cells were added to the pulse-labelled cells to serve as an internal control for recovery at various steps of sample manipulations. Using O'Farrell's twodimensional gel electrophoretic technique (O'Farrell, 1975) to separate the cellular proteins, the ratio of 3sS to 3H of each protein then was taken as the quantitative measurement of the relative rate of protein synthesis. The high resolution of O'Farrell's two-dimensional gel electrophoretic system made possible the direct examination of the decay rate of a large number of mRNAs coding for individual polypeptide chains. Materials and Methods Strains and Media. A haploid strain of Saccharomyces cerevisiae a ade 1 ade z ural tyrl his1 lys2 gall rnal), mutant ts136, was used in all experiments. Cultures were grown on a rotary shaker in media containing per liter: 6.7 g yeast nitrogen base minus amino acids (Difco), 21 mg each of all amino acids with the exception of methionine and cysteine, 100 mg adenine, 100 mg uracil, 20 g glucose, 200 mg each of yeast extract and of peptone, 10 g succihate, and 6 g sodium hydroxide. The doubling time was approximately 200 min. Cells were switched from 23 ° C to 37° C as follows: One half of the midlog phase culture was rapidly filtered through a 5 cm diameter Millipore filter (pore size 0.45 g) and the filtered media (without cells) was transferred to a 37° C water bath where 5 min was allowed for temperature equilibration. The second half of the midlog phase culture was filtered through another clean Millipore filter and then rapidly suspended in the prewarmed media described above. Although this procedure results in only half of the cells being transferred from 23 ° C to 37° C, it has the advantage of transferring cells from one temperature to another in a minimum length of time and into a preconditioned media having exactly the same composition.
Radioactive Labelling. Equal volumes (10 ml) of an exponentially growing ts136 culture were withdrawn at various intervals after the shift to 37° C and pulse-labelled for 5 min with 160 laCi [3~S]methionine (specific activity, 400 Ci/mmole from Amersham). The incorporation was linear over this labelling period. The pulses were terminated by adding cycloheximide (final concentration,
L.-L. Chia and C. McLaughlin: Half-Life of Yeast mRNA 0.5 mg/ml) and ice to the media. 3H-labelled cells were obtained from log phase culture labelled for 20 h with 3H-protein hydrolysate (30 gCi/ml). An equal volume (5 ml) of 3H-amino acid labelled cells was added to each of the pulse samples and used as an internal standard for recovery at various steps of sample manipulation.
Sample Preparation and Electrophoresis. Cells collected by centrifugation and pulse-labelled with [35S]methionine were grouped with cells labelled with 3H-amino acids and then resuspended in a small amount (< 100 gl) of ice cold water in a glass tube. To this was added 0.2 volumes of RNase-DNase solution (20 mM CaC12, 50 mM MgCI2, 0.5 mg/ml RNase, 1 mg/ml DNase, 0.5 M Tris, pH 7.0) and acid-washed glass beads (0.45 mm) level to the meniscus. The cells were lysed by vortexing three times for 30 s each at maximum speed on a vortex mixer. The tubes were cooled on ice between each vortexing. A 0.1 volume of RNase-DNase solution and a 0.1 volume of solution containing 0.1% sodium dodecyl sulfate (SDS) and 1% fi-mercaptoethanol were added. After 15 min, the sample was taken off ice and solid urea was added to a concentration of 9.5 M urea, followed by one volume of sample buffer containing 9.5 M urea, 4% Nonidet P-40 (w/v), 5% ~-mercaptoethanol, 2% ampholines (comprising 0.8% pH range 5 to 7, 0.8% pH range 5 to 8, and 0.4% pH range 3 to 10). The samples were maintained at room temperature and immediately loaded onto the gel. Two-Dimensional Gel Electrophoresis. Electrophoresis was performed according to the procedure of O'Farrell (1975) with the following modifications: The isoelectric focusing gel was 3% acrylamide and the sample overlay contained 9 M urea, 4% Nonidet P-40, 5% fi-mercaptoethanol, and 2% ampholines. The firstdimension gels were focused for 6,000 v-h. A 10% acrylamide gel with SDS was used for the second-dimension, The gels were stained, dried, and exposed to No-screen medical x-ray films according to standard procedures. To measure the radioactivity of a protein spot, the spot was cut from the dried gel and digested with 25 gl H20, 160 gl protosol and 4 ml toluene based scintillation fluid. The mixture was stored at room temperature with occasional shaking for three days before counting in a scintillation counter. The ratio of 3~S/3H counts in individual spots represented the relative rate of synthesis of each polypeptide. Polyribosome Analysis. Ribosomal fractions were prepared from a yeast whole cell culture by vortexing with glass beads followed by zone sedimentation in 10-40% (w/w) linear sucrose gradient in a Spinco model SW 56 ti rotor as previously described (Hartwell and McLaughlin, 1968).
Results Decay of Polyribosomes in ts136 at 37 ° C. The rapid
inhibition of RNA transport into the cytoplasm in mutant ts136 results in the decay of the mRNA in polyribosomes with the consequent conversion of polyribosomes into monoribosomes. Figure 1 shows the polyribosome profiles from whole cell cultures of the mutant incubated at 23° C and 37° C for 1 h, in which 71% and 12.8%, respectively, of the ribosomes were in polyribosomes. The decay of cytoplasmic polyribosomes in ts136 at 37° C is characteristic of the instability of existing mRNA. After the shift
L.-L. Chia and C. McLaughlin: Half-Life of Yeast m R N A
139
I
O
O
~
37 °
23° 3A
I
2 o
,.o (xl
1
," 70
45
m ©
"
.:
"1"
31 -
=~K)
i ". "
1~
7.~
4~
"
-
r~" '%
J
"75
25
77
-~ X ,,,=.4
C)
&
,
~
,~44
-,--
12 Fig. 4. Map of a yeast protein two-dimensional gel. This map was prepared from the autoradiogram of Figure 3A. Numbers were assigned to analyzed proteins. Molecular weight was determined by co-electrophoresis of bovine serum (67,000 daltons), ovalbumin (45,000 daltons), chymotrypsinogen (25,100 daltons), and hemoglobin (17,000 daItons
b .5 .2 t-~
II
-J
