Elucidating mechanisms of mitochondrial gene expression at nucleotide resolution

Harvard Medical School, Boston MA

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Abstract

DESCRIPTION (provided by applicant): Transcription and translation in mitochondria are fundamentally different from transcription and translation in eukaryotic nuclei and prokaryotic cells, and little is known in comparison. Regulation of mitochondrial gene expression, which must be sensitive to ever-changing energy needs and availability, is essential for maintaining proper levels of oxidative phosphorylation components. Balanced stoichiometry of these components is required to prevent production of harmful intermediates at reaction centers that cause aging phenotypes and disease. Steady-state mRNA levels of mitochondrial-encoded genes vary widely even though many transcripts are polycistronic, leading to the idea that regulation is primarily post-transcriptional. However there is growing evidence that in yeast RNA processing and translation are coupled with transcription. If this is the case, most regulation must take place co-transcriptionally. The extent of co-transcriptional regulation and the mechanisms by which it occurs are currently unknown because available techniques cannot easily differentiate between effects on transcription, processing, stabilization, and translation when they are tightly linked. This proposed work aims to adapt new, high-resolution techniques to study mitochondrial gene expression. First, native elongating transcript sequencing (NET-seq) and ribosome profiling will be established in this system to determine detailed, quantitative, genome-wide measures of transcription and translation. Following establishment of methods to analyze the datasets in conjunction, the combined NET-seq and ribosome profiling approach will be used to investigate the consequences of decoupling transcription, processing, and translation. Finally, the methodology will be applied to determine the primary mechanism of responding to changing cellular energy needs, and to monitor changes that happen with chronological aging in yeast. Ultimately, a detailed understanding of the mechanisms of mitochondrial gene regulation could be exploited in the design of new therapies to alleviate or prevent some of the effects of aging or the causes of some cancers and neurodegenerative diseases.