PPR family proteins in trypanosome mitochondria

描述（由申请人提供）：布鲁氏锥虫的线粒体基因表达在多个转录后水平受到调控，包括多顺反子前体加工、RNA编辑和RNA稳定性。尽管这些过程对于布鲁氏体生命周期中适当的基因表达至关重要，但控制其调控的反式作用因子在很大程度上是未知的。含五肽基序蛋白（PPRs）是最近发现的一类RNA结合蛋白，在叶绿体和线粒体中尤为丰富。在植物和真菌中，少数已被功能表征的PPRs主要参与特定细胞器rna的加工、稳定性和/或翻译。我们从现有的布鲁氏杆菌数据库中鉴定出6种含有多个PPR基序重复序列的蛋白质。这些蛋白质中的每一种在其n端也含有一个假定的线粒体靶向信号。我们的假设是，六个新发现的tbppr调节了布氏体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干扰来创建每个PPR蛋白缺失的细胞系，并确定PPR蛋白敲除对细胞生长和形态的影响。然后，我们将分析消耗每种PPR对特定线粒体rna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敲除细胞系中分离RNA，并评估1)使用northern blots检测RNA稳定性，2)使用毒化引物扩展进行RNA编辑，以及3)使用RT-PCR处理前体转录物。最后，我们将通过用GFP-PPR融合蛋白转染锥虫，确认6种布鲁氏体ppr蛋白的线粒体定位，并评估其中3种蛋白在细胞核上的潜在双重定位。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PPRs的功能在一般情况下还没有被很好地理解，它们可能也会为这个保守蛋白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.