Mitochondrial ribosomes and related RNP complexes in Trypanosoma brucei

描述（申请人提供）：锥虫线粒体翻译装置仍处于早期研究阶段。该系统使用模板进行操作，其中一些模板需要在翻译之前进行广泛编辑，可以预期该系统将与包括哺乳动物在内的其他系统中的系统完全不同。揭示和理解这些差异不仅是有趣的，而且还可以导致它们的治疗开发，以更好地治疗锥虫引起的疾病。最近的模式生物，利什曼原虫tarentolae的线粒体核糖体的分析，也证明了独特的异源二聚体45 S SSU* 复合物的presense代表的协会的核糖体小亚基与蛋白质复合物的性质知之甚少。这样的复杂性是前所未有的，其功能是未知的。这是可能的，这个复杂的是参与识别主管模板为随后的翻译，虽然没有数据来证实这一假设。本研究的目的是研究布氏锥虫线粒体核糖体及其相关复合物的存在，并采用RNA干扰技术敲低基因，以探讨45 S SSU* 复合物的潜在功能。选择这种生物体是因为它特别适合RNAi方法。 具体目标1：布氏锥虫线粒体核糖体RNP复合物的研究。线粒体核糖体核糖核蛋白（RNP）复合物的存在，主要是45 S SSU* 复合物，将在T。布鲁塞。将通过在蔗糖梯度中的速度沉降来分析纯化的线粒体的洗涤剂裂解物。将通过研究核糖体小亚基（9 S）和大亚基（12 S）核糖体RNA的沉降谱来检测核糖体复合物。复合物的身份将通过用抗体探测梯度级分来进一步研究，所述抗体将针对保守的小亚基和大亚基核糖体蛋白和推定的45 S SSU* 特异性蛋白产生。将对纯化的复合物45 S SSU* 进行质谱分析，以确定其在该物种中的蛋白质组成。 具体目标2：通过RNA干扰对45 S SSU* 复合物进行功能评价。45 S SSU* 复合物的功能将通过使用RNAi研究下调其几种蛋白质组分对复合物完整性的影响来探讨。靶点将包括几种45 S特异性蛋白，主要是在该复合物中发现的PPR蛋白。自RNAi诱导后7-14天内，每天研究靶mRNA和蛋白质水平，以估计敲除的功效以及生长动力学。在选定的时间点，将研究45 S SSU* 复合物以及单体和亚基的丰度（相对未指示细胞）。RNAi对线粒体翻译产物Cyb和COI从头合成的影响将通过研究35 S-掺入各自多肽的相对水平来研究。将通过蓝色非变性凝胶电泳研究对多肽稳定性及其组装成呼吸复合物的影响。 公共卫生相关性：布氏锥虫属于寄生原生生物，其引起影响全世界数百万人的疾病。目前对这些疾病的治疗并不令人满意。本项目旨在对T.布鲁氏菌线粒体核糖体和蛋白质合成系统的其他组成部分，在寄生虫的生命周期中发挥着不可或缺的作用。该系统具有作为化疗靶点的潜力。

DESCRIPTION (provided by applicant): Mitochondrial translation apparatus of trypanosomatids still is in the early investigation stage. This system operates with templates some of which need to be extensively edited prior to translation, one can anticipate that this system will be quite different from that in other systems including mammals. Revealing and understanding of these differences is not only interesting but can also lead to their therapeutic exploitation for better treatment of the diseases caused by trypanosomatids. The recent analysis of mitochondrial ribosomes in a model organism, Leishmania tarentolae, has also demonstrated the presense of unique heterodimeric 45S SSU* complexes representing an association of the ribosomal small subunit with a protein complex of a yet poorly understood nature. Such a complex is unprecedented and its function is unknown. It is possible that this complex is involved in recognition of competent templates for subsequent translation, although there are no data to substantiate this hypothesis. The goal of this proposal is to investigate the presence of mitochondrial ribosomes and the related complexes in in Trypanosoma brucei and to employ gene knock-down by RNA interference in order to probe into potential functions of the 45S SSU* complexes. This organism is chosen because it is particularly amenable to the RNAi approach. Specific aim 1: Investigation of mitochondrial ribosomal RNP complexes in Trypanosoma brucei. The existence of mitochondrial ribosomal ribonucleoprotein (RNP) complexes, primarily 45S SSU* complexes, will be investigated in T. brucei. Detergent lysates of purified mitochondria will be analyzed by velocity sedimentation in sucrose gradients. Ribosomal complexes will be detected by investigating sedimentation profiles of the ribosomal small subunit (9S) and large subunit (12S) ribosomal RNAs. The identity of the complexes will be further investigated by probing gradient fractions with antibodies that will be produced against conserved small subunit and large subunit ribosomal proteins and presumtive 45S SSU*-specific proteins. A mass-spectroscopy analysis of the purified complexes 45S SSU* will be performed to ascertain their protein composition in this species. Specific aim 2: Functional evaluation of the 45S SSU* complexes by RNA interference. The function of the 45S SSU* complex will be probed into by investigating the impact of down-regulating several of its protein components on the integrity of the complexes using RNAi. The targets will include several 45S-specific proteins, mainly the PPR proteins found in this complex. The target mRNA and protein levels will be investigated daily over the period of 7-14 days since the induction of RNAi to estimate the efficacy of the knock-downs, in addition to growth kinetics. At a selected time point(s), the abundance (relative unindiced cell) of the 45S SSU* complexes, as well as monosomes and subunits, will be investigated. The effect of the RNAi on the de novo synthesis of the mitochondrial translation products Cyb and COI will be investigated by studying relative levels of 35S-incorporation into the respective polypeptides. The effect on the stability of the polypeptides and their assembly into the respiratory complexes will be studied by Blue Native gel electrophoresis. PUBLIC HEALTH RELEVANCE: Trypanosoma brucei belongs to the group of parasitic protists which cause the diseases affecting millions of people worldwide. The current treatments of these diseases are not satisfactory. The project is aimed at investigation of T. brucei mitochondrial ribosomes and other components of the protein synthesis system that plays an indispensable role during the parasite's life cycle. This system has a potential as a target for chemotherapy.