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Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Published on: March 10, 2012
Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component
Kelly Salinas1, Sara Wierzbicki, Li Zhou
1Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Abstract:
In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.
Insights
Researchers identified a new protein component, YLR145w (now RMP1), essential for the mitochondrial RNA processing (MRP) complex in yeast. This discovery clarifies MRP
Area of Science:
- Molecular biology
- Yeast genetics
- RNA processing
Background:
- RNase mitochondrial RNA processing (MRP) is crucial in Saccharomyces cerevisiae.
- RNase MRP shares eight protein components with RNase P, complicating characterization.
- Previous studies lacked a complete understanding of RNase MRP's protein composition.
Purpose of the Study:
- To fully characterize the protein components of the RNase MRP complex.
- To identify novel proteins associated with RNase MRP.
- To determine the specific roles of newly identified proteins in RNA processing.
Main Methods:
- Purification of RNase MRP using tandem affinity purification.
- Co-precipitation assays to identify interacting proteins.
- Construction and analysis of a temperature-sensitive ylr145w mutant.
Main Results:
- Tandem affinity purification yielded a highly active RNase MRP complex.
- YLR145w and Rpr2 were identified as additional protein components of RNase MRP.
- YLR145w specifically co-precipitated with RNase MRP RNA, not RNase P RNA.
- A ylr145w mutant exhibited rRNA processing defects, but not tRNA processing defects.
- Homologues of YLR145w are conserved in various eukaryotes, including plants.
Conclusions:
- YLR145w encodes a novel protein component of RNase MRP, essential for its function.
- YLR145w is not a component of RNase P.
- The gene YLR145w is proposed to be renamed RMP1 (RNase MRP protein 1).
- This finding advances the understanding of RNase MRP structure and function.
