PPR family proteins in trypanosome mitochondria

描述(申请人提供)：布鲁氏锥虫线粒体基因表达在多转录后水平上受到调控，包括多顺反子前体处理、RNA编辑和RNA稳定性。虽然这些过程对于布氏毛滴虫生活史中适当的基因表达是至关重要的，但控制它们的反式作用因素在很大程度上是未知的。五肽基序包含蛋白(Pprs)是最近发现的一个RNA结合蛋白家族，特别丰富的是叶绿体和线粒体。少数已被确定功能的PPR主要参与植物和真菌中特定细胞器RNA的加工、稳定和/或翻译。我们在现有的布鲁氏锥虫数据库中鉴定了六种含有多个PPR基序重复的蛋白质。这些蛋白质中的每一个都在其N端含有一个假定的线粒体靶向信号。我们的假设是，这六个新发现的TbPPR调控布鲁氏毛滴虫线粒体中特定的转录后基因调控事件。为了验证这一假设，我们将使用RNA干扰创建在每个PPR中耗尽的细胞系，并确定PPR蛋白敲除对细胞生长和形态的影响。然后，我们将分析耗尽每个PPR对特定线粒体RNA的加工和/或稳定性的功能后果。为此，我们将从未诱导和诱导的击倒细胞系中分离RNA，并使用Northern blotts评估1)RNA稳定性，2)使用有毒引物延伸进行RNA编辑，以及3)通过RT-PCR对前体转录进行处理。最后，我们将确认6个布氏锥虫PPR的线粒体定位，并通过将GFP-PPR融合蛋白导入锥体来评估其中3个蛋白质对核的双重定位的可能性。这些研究将为锥虫线粒体基因调控机制提供重要的新信息。由于PPR的功能一般还没有被很好地理解，它们也可能为这个保守的蛋白质家族在高等生物中的功能提供新的见解。

DESCRIPTION (provided by the applicant): Mitochondrial gene expression in Trypanosoma brucei is regulated at multiple posttranscriptional levels including polycistronic precursor processing, RNA editing, and RNA stability. Although these processes are critical for proper gene expression during the T. brucei life cycle, the trans-acting factors that govern their regulation are largely unknown. Pentatricopeptide motif containing proteins (PPRs) comprise a recently described family of RNA binding proteins that are particularly abundant chloroplasts and mitochondria. The few PPRs that have been functionally characterized are primarily involved in the processing, stability, and/or translation of specific organellar RNAs in plants and fungi. We identified six proteins containing multiple PPR motif repeats in the available T. brucei databases. Each of these proteins also contains a putative mitochondrial-targeting signal at its N-terminus. Our hypothesis is that the six newly identified TbPPRs modulate specific posttranscriptional gene regulatory events in T. brucei mitochondria. To test this hypothesis, we will create cell lines depleted in each of the PPRs using RNA interference and determine the effect of PPR protein knock-down on cell growth and morphology. We will then analyze the functional consequences of depleting each PPR on the processing and/or stability of specific mitochondrial RNAs. To this end, we will isolate RNA from un-induced and induced knock-down cell lines and assess 1) RNA stability using northern blots, 2) RNA editing using poisoned primer extension, and 3) precursor transcript processing by RT-PCR. Finally, we will confirm the mitochondrial localization of each of the six T. brucei PPRs and assess the potential dual localization of three of the proteins to nuclei by transfection of trypanosomes with GFP-PPR fusion proteins. These studies will provide significant new information regarding the mechanisms of mitochondrial gene regulation in trypanosomes. Since the functions of PPRs are not well understood in general, they will likely provide novel insights into the functions of this conserved protein family in higher organisms as well.