The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.
mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell’s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.
The transcription and decay of specific mRNA are crucial determinants of gene expression. The rate of synthesis and decay of specific mRNAs determines the steady-state level of that particular mRNA. The steady state levels of mRNAs govern the abundance of mRNAs and determine how much of each mRNA is available for protein synthesis. Measurements of mRNA half-lives are used extensively to determine the decay rate of mRNAs. Specific mRNAs decay at different rates that are related to features of the mRNA, the function of the protein encoded by the mRNA and the environmental conditions. Depending on the technique utilized to determine mRNA decay rates, decay rate measurements can be determined either globally or for individual transcripts. In the yeast S. cerevisiae, the techniques that are most commonly used to measure global mRNA decay rates include utilizing a yeast strain harboring the temperature sensitive allele of RNA polymerase II and chemical transcriptional inhibitors such as thiolutin and 1,10-phenanthroline 1-5. These methods can also be utilized to measure individual mRNA decay rates 4. Other methods can also be utilized to measure mRNA decay rates. These methods include approach to steady state labeling or utilization of mRNA molecules that are expressed from a regulated promoter that is expressed only in select conditions. Each of these techniques has certain advantages and limitations. The technique described here utilizes the temperature sensitive allele of RNA polymerase II. This method uses S. cerevisiae as the model, but can been modified and utilized in other systems using specific transcriptional inhibition techniques 6.
mRNA half-life measurements using the temperature sensitive allele of RNA polymerase II are extensively used for both genome-wide and individual measurements of mRNA decay 4-5. This technique requires the use of a specific yeast strain that harbors a temperature sensitive allele of RNA polymerase II, rpb1-11. The rationale for this technique is that exposure of the temperature sensitive yeast strain to the nonpermissive temperature inhibits mRNA synthesis. Subsequently, decay of the preexisting mRNA is monitored at different time points after transcription has been inhibited. The disappearance of the preexisting mRNA is monitored by extracting RNA from the yeast cells at different time points after transcription has been turned off. The time points at which the yeast cells are harvested are predetermined by a pilot experiment, and depend on the transcripts and system being investigated. Quicker time points are used for short-lived transcripts, while longer time points are used for longer lived transcripts. Afterwards, the decay of the mRNA is monitored by either northern blot analysis, quantitative PCR or RNAseq.
Measurement of mRNA half-lives using a temperature sensitive allele of RNA polymerase II has its advantages. First, this technique is easy and straight forward. Second, once the yeast strain is acquired or generated in the laboratory the mRNA half-life measurements can be determined in different growth conditions; enabling determination of environmental influence on mRNA decay. Third, mRNA decay rates can be monitored genome-wide. Use of other transcription inhibition techniques also has advantages and limitations. For example, use of an inducible promoter requires subcloning to generate an mRNA that is under the control of the regulated promoter. Thiolutin is not readily available and is expensive when available. In addition, thiolutin’s mode of action is not completely understood and it has been reported to affect other cellular processes including inhibiting mRNA decay 7. Alternatively, 1,10-phenanthroline, is more readily available. Furthermore, all of the techniques used to inhibit transcription can perturb cellular function and can affect different mRNAs in distinct ways. An investigator needs to determine the most appropriate method to use in their experimental conditions to attain the most reliable results. To determine which method is most suitable for their application, a researcher needs to identify the transcripts and function of the proteins encoded by the transcripts being investigated. The most reliable mRNA decay rate measurements are those that are determined using multiple techniques and show the same decay rate. No single technique is always the best, and the most appropriate technique depends on the specific situation.
Numerous studies in S. cerevisiae have measured mRNA decay rates in various conditions and genetic backgrounds. The conditions that mRNA decay rates are measured in depend on the specific experiment being investigated. Measuring mRNA decay rates in different cellular environments determines whether the conditions being examined preferentially affect the decay rates of specific mRNAs. The decay rates of mRNAs can also vary depending on the yeast strain being used. For example, mRNA decay rates can be determined in wild-type yeast cells and yeast cells with a nonfunctional nonsense-mediated mRNA degradation (NMD) pathway. This mRNA degradation pathway is found in all eukaryotic organisms that have been examined so far and it triggers the degradation of mRNAs that prematurely terminate translation 8. NMD was initially identified as a pathway that degrades mRNAs with premature termination codons or nonsense codons, but is now recognized as a pathway that also regulates the expression of non-nonsense containing natural mRNAs. mRNAs that are targets of the pathway are rapidly degraded in yeast cells with a functional NMD pathway and stabilized in yeast cells with a nonfunctional NMD pathway. Thus, the half-lives of mRNAs that are direct targets of this pathway are shorter in wild-type yeast cells compared to yeast cells with a nonfunctional NMD pathway.
1. Growth of Yeast Cells
2. Harvest the Yeast Cells
3. Extract RNA from the Yeast Cells
4. Northern Blot Analysis
NOTE: Use northern blots to quantify mRNA levels and obtain information on the size of the transcripts. In addition, use northern blots to detect mRNAs that produce multiple isoforms of the same mRNA.
5. Hybridize Probes Complementary to the RNA of Interest to the Membrane
NOTE: One way to detect mRNA on the membrane is to hybridize 32P labeled DNA probes. CAUTION: Researchers working with 32P need to use protective procedures to prevent contamination. Follow institutional guidelines on use of radioactive material.
6. Quantify the RNA that is Bound to the Membrane
NOTE: To quantify the amount of radioactivity on membrane, expose the membrane to a Phosphor screen. After the appropriate exposure time, scan the Phosphor screen using a phosphorimager.
The ability of this protocol to accurately measure mRNA decay rates depends on inhibition of transcription, the harvesting of yeast cells at the appropriate time points, and utilization of RNase free techniques while extracting RNA and northern blotting. Probing for two control mRNAs known to be unstable and stable, respectively, provides confidence that the experiment worked. For example, this can be accomplished by probing with a probe that detects both the CYH2 pre-mRNA and mRNA. Figure 2B shows the disappearance of the CYH2 pre-mRNA in wild-type and nmd mutant yeast strains at different time points after transcription inhibition. The CYH2 pre-mRNA is degraded faster in yeast cells with a functional NMD pathway (UPF1) relative to yeast cells with a nonfunctional NMD pathway (upf1).
Figure 1. Method flowchart. Measurement of mRNA decay rate flowchart
Figure 2. mRNA half-life of CYH2-pre-mRNA and mRNA. (A) Schematic representation of the CYH2 pre-mRNA and CYH2 mRNA. CYH2 pre-mRNA is inefficiently spliced and transported to the cytoplasm where it is degraded by the NMD pathway. The CYH2 mRNA is not degraded by the NMD pathway because it lacks the intron containing the premature termination codon (PTC). (B) Half-life northern blots of RNA extracted from wild-type (UPF1) and nmd mutant strains (upf1). The time points after transcription inhibition are listed above the northern blots. The blots were probed with radiolabelled CYH2 DNA. (C) A graph of % CYH2 pre-mRNA remaining versus time in wild-type (UPF1) and nmd mutant strains (upf1). This graph shows that the CYH2 pre-mRNA is degraded at a faster rate in wild-type (UPF1) than in nmd mutant yeast strains (upf1).
Inhibition of mRNA synthesis and monitoring mRNA turnover in the absence of new synthesis is a method that is frequently used to measure mRNA decay rates. In S. cerevisiae, measurement of mRNA decay rates by inhibiting transcription using the temperature sensitive allele of RNA polymerase II is one of the most frequently used methods. This method specifically inhibits RNA polymerase II. The most critical steps for determination of mRNA decay rates using this technique are: 1) Prior to harvesting the yeast cells, ensure that transcription has been shut off by maintaining the culture at 39°C; 2) During the RNA extraction and RNA gel electrophoresis steps of the protocol ensure that RNase free techniques are used; 3) Use a control RNA for normalization to ensure that equal amounts of RNA were loaded and that the experimental treatments are working as expected; 4) repeat the mRNA decay rate measurements at least three times to ensure reproducibility and accuracy of the half-life measurements.
rpb1-1 yeast strains are normally transferred to 37 °C to inhibit transcription. However, we have found that at 37 °C inhibition of transcription occurs ~3 min after the temperature shift. At 39 °C transcription inhibition is immediate 4, 11. However, the use of this technique to determine mRNA decay rates has some limitations. The primary limitation is that a special yeast strain is required. As previously stated, this yeast strain can either be obtained from other laboratories or generated in the laboratory if a specific yeast genetic background is required. Once the yeast strain is obtained, this technique is simple and straight forward. A second limitation is that the method entails exposing the yeast cells to heat shock to inhibit transcription. Heat stress can affect cellular processes including the decay rate of particular mRNAs. For example, the decay rate of those mRNAs that encode for proteins that are involved in stress response may be affected. Lastly, the utilization of a yeast strain with a mutation in RNA polymerase II can result in the production of alternative transcripts that behave differently from the normal transcripts.
As discussed in the introduction, other techniques are utilized to measure mRNA decay rates in S. cerevisiae. This includes inhibition of transcription using chemicals such as thiolutin and 1-10-phenanthroline. These techniques are advantageous in that they can be done using any yeast strain and mRNA decay rates can also be determined genome-wide or for individual endogenous transcripts. In addition, mRNA decay rate measurements can be done in various physiological conditions. The utility of these drugs is limited by the fact that they can also affect cellular processes and influence the decay rates of mRNA differentially. Additionally, thiolutin is not readily available and when available is expensive.
After mastering this technique one will be able to determine mRNA decay rates of individual mRNAs or genome-wide. In addition, mRNA decay rates can be measured in different physiological conditions to examine whether different conditions affect mRNA decay rates differentially.
The authors have nothing to disclose.
Research in the author’s laboratory is supported by the Texas Higher Education Coordinating Board’s Norman Hackerman Advanced Research Program and start-up funds from Baylor University.
Name of Material/Equipment | Company | Catalog Number | Comments/Description |
High Speed Centrifuge | Eppendorf | 22628169 | |
Mini Centrifuge | Fisher Scientific | 05-090-100 | |
Genescreen Plus membrane | PerkinElmer | 50-905-0169 | |
Hybridization Oven | Fisher Scientific | 95-0030-01 | |
Nanodrop spectrophotometer | Thermo Scientific | ND-8000 | |
Phosphor screen | GE Healthcare Life Sciences | 28-9564-78 | |
Typhoon phosphorimager | GE Healthcare Life Sciences | 29004080 | |
UV cross-linker | GE Healthcare Life Sciences | 80-6222-31 | Alternatively the membrane can be baked in an oven set to 80° for 1 hour |
NorthernMax prehybridization/hybridization buffer | Life Technologies | AM8677 | |
Yeast strains harboring the rpb1-1 mutation | Yeast strains can be obtained from a laboratory or created with a specific background |