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核和原核细胞中的转录和翻译根本不同，并且在比较中知之甚少。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，对于维持氧化磷酸化组分的适当水平至关重要。需要这些组分的平衡化学计量，以防止在反应中心产生导致老化表型和疾病的有害中间体。 尽管许多转录物是多顺反子的，但mRNA水平的稳态差异很大，这导致了调控主要是转录后的观点。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相结合。如果是这样的话，大多数调控必须以共转录的方式进行。共转录调控的程度及其发生的机制目前尚不清楚，因为现有技术无法轻易区分对转录、加工、稳定和翻译的影响（当它们紧密相连时）。这项拟议的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，将在该系统中建立天然延伸转录物测序（NET-seq）和核糖体分析，以确定详细的、定量的、 转录和翻译的全基因组测量。在建立了结合分析数据集的方法之后，将使用组合的NET-seq和核糖体分析方法来研究解耦转录、加工和翻译的后果。最后，该方法将被应用于确定响应不断变化的细胞能量需求的主要机制，并监测酵母中随时间老化发生的变化。 最终，对线粒体基因调控机制的详细了解可以用于设计新疗法，以减轻或预防衰老的一些影响或某些癌症和神经退行性疾病的原因。

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.