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Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains
Published on: December 13, 2014
A nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae
Letian Kuai1, Biswadip Das, Fred Sherman
1Department of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, Rochester, NY 14642, USA.
Abstract:
We previously demonstrated an increased degradation of mRNAs in mutants of Saccharomyces cerevisiae having blocks in nuclear export. The degradation activity, designated DRN (degradation of mRNA in the nucleus), requires Cbc1p, a nuclear cap-binding protein, and Rrp6p, a nuclear exosome component. Microarray procedures were used to determine the half-lives of mRNAs from normal and mutant strains, leading to the tentative identification of hundreds of normal mRNAs that were notably stabilized when either CBC1 or RRP6 were deleted. Northern blot analysis of representative mRNAs confirmed the diminished degradation. One representative of this group, SKS1 mRNA, was also shown by a cytological procedure to be preferentially retained in the nucleus compared with typical mRNAs. We suggest that all normal mRNAs are subjected to degradation by DRN, but the degree of degradation is determined by the degree of nuclear retention. Furthermore, these mRNAs particularly susceptible to DRN were also diminished by overproduction of Cbc1p, demonstrating a regulatory role for CBC1. This conclusion was corroborated by finding an inverse relationship of the CBC1 and SKS1 mRNA levels in normal strains grown under different conditions.
Insights
Nuclear export defects in yeast increase mRNA degradation via Cbc1p and Rrp6p. This degradation, termed DRN, targets nuclear-retained mRNAs, with Cbc1p playing a key regulatory role.
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Metabolism
Background:
- Nuclear export is crucial for mRNA processing and function.
- Defects in nuclear export can lead to altered mRNA stability.
- The Saccharomyces cerevisiae model system is used to study fundamental cellular processes.
Purpose of the Study:
- To investigate the mechanism of increased mRNA degradation in yeast mutants with nuclear export blocks.
- To identify the factors involved in this degradation process, designated DRN (degradation of mRNA in the nucleus).
- To elucidate the regulatory role of Cbc1p and Rrp6p in mRNA turnover.
Main Methods:
- Microarray analysis to determine mRNA half-lives in wild-type and mutant yeast strains.
- Northern blot analysis to validate degradation patterns of specific mRNAs.
- Cytological methods to assess mRNA localization within the nucleus.
Main Results:
- Deletion of CBC1 or RRP6 significantly stabilized hundreds of normal mRNAs, indicating their role in degradation.
- SKS1 mRNA, a representative target, showed preferential nuclear retention and diminished degradation upon CBC1 or RRP6 deletion.
- Overproduction of Cbc1p diminished mRNAs susceptible to DRN, confirming its regulatory function.
Conclusions:
- The DRN pathway, requiring Cbc1p and Rrp6p, degrades nuclear-retained mRNAs in Saccharomyces cerevisiae.
- Nuclear retention correlates with susceptibility to DRN-mediated degradation.
- Cbc1p plays a critical regulatory role in controlling mRNA degradation by DRN.
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