PPR family proteins in trypanosome mitochondria

描述（由申请人提供）：布氏锥虫中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T.尽管布鲁氏菌的生命周期中存在着许多问题，但支配其调节的反式作用因素在很大程度上是未知的。含有五肽基序的蛋白质（PPRs）包括最近描述的在叶绿体和线粒体中特别丰富的RNA结合蛋白家族。已经被功能表征的少数PPR主要参与植物和真菌中特定细胞器RNA的加工、稳定性和/或翻译。我们在T. brucei数据库。这些蛋白质中的每一种在其N-末端还含有推定的神经靶向信号。我们的假设是，六个新发现的TbPPRs调节T.布鲁氏菌线粒体。为了检验这一假设，我们将使用RNA干扰建立每个PPR缺失的细胞系，并确定PPR蛋白敲低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敲低细胞系中分离RNA，并评估1）使用北方印迹的RNA稳定性，2）使用中毒引物延伸的RNA编辑，和3）通过RT-PCR的前体转录物加工。最后，我们将确认六个T中的每一个的线粒体定位。通过用GFP-PPR融合蛋白转染锥虫，评估三种蛋白质在细胞核中的潜在双重定位。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的功能一般还没有很好地理解，它们可能会提供新的见解，这个保守的蛋白质家族在高等生物中的功能。

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.