Elucidating mechanisms of mitochondrial gene expression at nucleotide resolution

描述(申请人提供)：线粒体中的转录和翻译与真核和原核细胞中的转录和翻译有根本的不同，比较起来知之甚少。对线粒体基因表达的调节必须对不断变化的能量需求和可获得性很敏感，这对于维持适当水平的氧化磷酸化成分至关重要。这些成分的平衡化学计量比是必需的，以防止反应中心产生有害的中间体，从而导致衰老表型和疾病。尽管许多转录本是多顺反子的，但线粒体编码基因的稳态mRNA水平差异很大，这导致了调控主要是转录后调控的想法。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，大多数监管必须以协同转录的方式进行。共转录调控的程度和发生机制目前尚不清楚，因为现有的技术不容易区分对转录、加工、稳定和翻译的影响，当它们紧密联系在一起时。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的表达。首先，将在该系统中建立天然延长转录本测序(NET-SEQ)和核糖体图谱，以确定详细的、定量的、 全基因组的转录和翻译措施。在建立了联合分析数据集的方法之后，将使用Net-Seq和核糖体图谱相结合的方法来调查分离转录、加工和翻译的后果。最后，该方法将被应用于确定对不断变化的细胞能量需求做出反应的主要机制，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被用来设计新的疗法，以减轻或防止衰老的一些影响，或者一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.