Elucidating mechanisms of mitochondrial gene expression at nucleotide resolution

描述（由申请人提供）：线粒体中的转录和翻译与真核细胞和原核细胞中的转录和翻译有着根本的不同，相比之下我们所知甚少。线粒体基因表达的调节必须对不断变化的能量需求和可用性敏感，对于维持适当的氧化磷酸化成分水平至关重要。需要平衡这些成分的化学计量，以防止在反应中心产生导致衰老表型和疾病的有害中间体。线粒体编码基因的稳态mRNA水平变化很大，尽管许多转录本是多顺反子的，这导致了调控主要是转录后的想法。然而，越来越多的证据表明，在酵母中，RNA的加工和翻译与转录是耦合的。如果是这样的话，大多数调控一定是协同转录的。共转录调控的程度及其发生的机制目前尚不清楚，因为现有的技术不能轻易区分当它们紧密相连时对转录、加工、稳定和翻译的影响。这项工作旨在采用新的高分辨率技术来研究线粒体基因表达。首先，本系统将建立原生延长转录序列（NET-seq）和核糖体分析，以确定详细的、定量的、

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.