Title: Import of RNA into Mitochondria. The main goal of the research outlined in this proposal is to understand how an RNA molecule is imported into the mitochondrial compartment and to identify the machinery involved in this process. As a model system we have been using the Saccharomyces cerevisiae RNase MRP, a ribonucleoprotein endoribonuclease that has at least two roles in cellular RNA processing, one in the mitochondrial compartment where it plays a direct role in the initiation of mitochondrial DNA replication and a second in the nucleolus. The specific objective for this proposal is to understand the mechanism and pathway by which the RNA subunit of the ribonucleoprotein enzyme RNase MRP is imported into mitochondria. Loss of mitochondrial DNA through mutation, deletion or depletion has been implicated in heart disease, diabetes, muscle myopathies, and more recently aging. Understanding the role of RNase MRP in mitochondrial DNA replication and function may provide better treatments for patients with these diseases. Understanding how RNAs are imported into mitochondria would provide a mode of gene therapy for people with mitochondrial DNA mutations without the need to transform mitochondria. Experimentation is based on the rationale that the genes for the RNA component of RNase MRP is nuclear encoded; hence, the MRP RNA needs to be transported from its site of transcription in the nucleus to mitochondria and be assembled with its cognate proteins. The RNase MRP enzyme in the yeast offers an ideal system to study RNA import into the mitochondrion using both biochemical and genetic methodologies.
The specific aims enumerated below are designed to use both biochemistry and genetics to identify the mechanism and machinery of RNA import into mitochondria.
Specific aims will include. 1) Use of a newly developed in vitro RNA import system to elucidate the biochemical and molecular requirements for RNA import. 2) Isolation of mutations in the MRP RNA that are specific for mitochondrial import to determine the sequences required for mitochondrial substrate binding, mitochondrial specific protein binding and import into the mitochondrial matrix. 3) Characterization of these mutants for various aspects of mitochondrial metabolism including loss of mitochondrial respiration, DNA maintenance, translation, processing of RNAs and import of the mutant MRP RNA in our in vitro system. 4) Identification of other RNA molecules imported into mitochondria. These experiments should provide a wealth of knowledge about this fundamental process.
| Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S et al. (2010) Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components. RNA 16:529-37 |
| Salinas, Kelly; Wierzbicki, Sara; Zhou, Li et al. (2005) Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component. J Biol Chem 280:11352-60 |
