Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Summary

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.

Abstract

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.

Introduction

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 measureme....

Protocol

1. Growth of Yeast Cells

1. Select the appropriate yeast strains to be utilized for the mRNA decay rate measurements. To inhibit transcription using the temperature sensitive allele of RNA polymerase II, use yeast strains harboring the rpb1-1 mutation ¹. Obtain this yeast strain from a laboratory that already has one, or generate it in the laboratory using standard techniques if a specific genetic background is required⁹.
2. Using sterile technique, prepare yeast media using standard procedures ¹⁰. If no selection is required, prepare rich media such as YPD. Alternatively, prepare selective media if the yeast c....

Results

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 sh.......

Discussion

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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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