Leveraging genomic approaches to characterize mitochondrial RNA biology
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CitationWolf, Ashley Robin. 2014. Leveraging genomic approaches to characterize mitochondrial RNA biology. Doctoral dissertation, Harvard University.
AbstractTranscription and translation of mammalian mitochondrial DNA (mtDNA) occurs within the mitochondrial matrix to produce oxidative phosphorylation subunits required for efficient energy production. These mtDNA-encoded subunits complex with mitochondrial-localized, nuclear-encoded subunits to form the respiratory chain, and aberrant production or function of these subunits can cause devastating human disease. In addition to 13 oxidative phosphorylation subunits, mtDNA encodes 2 rRNAs and 22 tRNAs. All proteins required for mitochondrial RNA transcription, processing, and translation are encoded in the nucleus and translocated into the mitochondria. Here, I characterize over 100 nuclear-encoded mitochondrial proteins with predicted RNA-binding domains. Using RNAi and an RNA profiling approach, MitoString, we further characterize previously identified RNA processing factors and identify the novel regulator FASTKD4, which influences the abundance of a subset of mitochondrial mRNAs. Next, we apply knowledge of the RNA degradation component SUPV3L1 gleaned from our RNAi studies and previous research to test whether a specific set of variants influence the function of this gene in patient fibroblasts. Using MitoString, we find no evidence of pathogenicity of these variants in our fibroblast model. Our approach highlights the value of a thorough understanding of mitochondrial proteins and the necessity of experimental techniques to validate the effect of variants found in exome-sequencing studies. Finally, we take an unbiased approach to characterizing the mitochondrial transcriptome of mouse liver by sequencing RNA from sequentially enriched mitochondrial fractions. Although we find an abundance of nuclear-encoded 5S rRNA, consistent with previous research, we fail to identify any imported nuclear-encoded tRNAs. Uniting genomics, biochemistry, and medicine, these findings advance our understanding of mitochondrial RNA biology.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12274280
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