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Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
Published on: March 14, 2014
Scp160p associates with specific mRNAs in yeast
Ai-Min Li1, Alice Watson, Judith L Fridovich-Keil
1Department of Human Genetics, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.
Abstract:
Scp160p is a multiple KH-domain RNA-binding protein in yeast that has been demonstrated previously to associate with both soluble and membrane-bound polyribosomes as an mRNP component. One key question that has remained unanswered, however, is whether the mRNAs in these mRNP complexes are random or specific. We have addressed this question using microarray analyses of RNAs released from affinity isolated Scp160p-containing complexes, compared with total RNA controls from the same lysates. Our results, confirmed by quantitative RT-PCR analysis, clearly demonstrate that Scp160p associates with specific rather than with random messages, and that among the enriched targets are DHH1, YOR338W, BIK1, YOL155C and NAM8. Furthermore, loss of Scp160p resulted in a significant change in both the abundance and distribution between soluble and membrane-associated fractions for at least one of these messages (YOR338W), and in a subtle yet significant shift from soluble polyribosomes to soluble mRNPs for at least two of these target messages (DHH1 and YOR338W). Together, these data not only identify specific mRNA targets associated with Scp160p in vivo, they demonstrate that the association of Scp160p with these messages is biologically relevant.
Insights
The yeast protein Scp160p binds specific messenger RNAs (mRNAs), not random ones. Loss of Scp160p affects mRNA abundance and localization, showing its biological relevance in regulating gene expression.
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Biology
Background:
- Scp160p is a yeast protein known to bind messenger RNA (mRNA) on polyribosomes.
- It remains unclear if Scp160p associates with specific or random mRNAs.
Purpose of the Study:
- To determine if Scp160p binds specific mRNAs.
- To investigate the biological relevance of Scp160p-mRNA interactions.
Main Methods:
- Affinity isolation of Scp160p-containing complexes.
- Microarray analysis of associated RNAs.
- Quantitative RT-PCR validation.
- Analysis of mRNA distribution upon Scp160p loss.
Main Results:
- Scp160p specifically associates with certain mRNAs, including DHH1, YOR338W, BIK1, YOL155C, and NAM8.
- Loss of Scp160p alters the abundance and localization of YOR338W mRNA.
- Scp160p depletion causes a shift in DHH1 and YOR338W mRNA from polyribosomes to mRNPs.
Conclusions:
- Scp160p is a specific RNA-binding protein in yeast.
- Scp160p plays a biologically relevant role in mRNA regulation and localization.
