SMALL RNAS AND THEIR ROLE IN TRYPANOSOME BIOLOGY

描述(申请人提供)：非洲锥虫在撒哈拉以南非洲的广大地区造成疾病和痛苦，至少可以说，可用于消除这种疾病的药物是次优的。许多实验室，包括我们自己的实验室，一直致力于研究布鲁氏锥虫的生物学，并为了解锥虫特有的几种机制做出了贡献。锥虫特异途径的发现是确定新的潜在药物靶点的第一步，这些靶标可能被用来对抗寄生虫本身。自1998年我们在布氏锥虫中发现RNAi途径以来，我们的研究一直集中在了解RNAi的机制和组成上。这些研究对于最终尝试将小RNA用作对抗锥虫的药物至关重要。这种可能性并不是没有根据的，因为已经有几项临床试验使用小RNA来治疗某些人类疾病。我们提出的实验直接源于上一个资助期内产生的三个主要结果。首先，对来自血流和前循环锥体的小干扰RNA(SiRNAs)进行了测序，揭示了RNAi途径的一个新靶点，即位于染色体内部位点的卫星样重复序列家族(CIR147)。这些重复序列可能在染色体结构和/或基因表达调控的某些方面发挥作用。其次，我们已经在巴西利什曼原虫中鉴定了RNAi基因，并与Steve Beverley合作证明了RNAi途径是可操作的。然而，由于目前尚无合适的小动物模型，L在研究发病机制方面存在一定的局限性。相比之下，耶鲁大学的戴安娜·麦克马洪-普雷特为泛美乳杆菌建立了一个小鼠模型，泛美乳杆菌是巴西乳杆菌的近亲，也具有RNAi基因。因此，我们的发现为建立工具，将RNAi技术的奇迹应用于利什曼原虫生物学和发病机制的不同方面的研究开辟了道路。第三，我们发现第二个布鲁氏锥虫类蛋白(称为PiwiS)定位于线粒体中，并形成了使人想起一些运动基质DNA(KDNA)复制酶定位的反足部位的焦点，暗示PiwiS在kDNA生物发生的某些方面。在过去的几年里，在转录和转录后水平上，ArgAerte蛋白已经成为一系列现象的中心调节者。因此，我们假设PiwiS在线粒体RNA沉默的某些方面发挥作用。应该注意的是，这种蛋白质是第一个定位于细胞器的ArgAerte蛋白质的例子。我们在下一个资助期的具体目标是：1.通过对布鲁氏锥虫和巴西锥虫的小RNA进行高通量测序，以及通过CIR147卫星样重复序列家族的功能和结构特征，进一步挖掘锥虫的生物学。2.建立可遗传的、可调控的泛美白对虾RNAi工具。3.建立PiwiS在布氏毛滴虫线粒体中的功能。 项目简介：非洲锥虫在撒哈拉以南非洲的广大地区造成疾病和痛苦，至少可以说，可用于消除这种疾病的药物是次优的。我们的研究重点是了解RNA干扰的机制和成分，最终试图将小RNA用作对抗锥虫的药物。这种可能性并不是没有根据的，因为已经有几项临床试验使用小RNA来治疗某些人类疾病。

DESCRIPTION (provided by applicant): African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Many laboratories, including our own, have been engaged in studying the biology of Trypanosoma brucei and have contributed to the understanding of several mechanisms that are unique to trypanosomes. The discovery of trypanosome-specific pathways is the first step towards identifying new potential drug targets, which may be exploited against the parasite itself. Since 1998 when we discovered the RNAi pathway in Trypanosoma brucei our research has focused towards understanding the mechanism and components of RNAi. These studies are essential to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases. The experiments we propose stem directly from three main findings generated during the last funding period. First, sequencing of small interfering RNAs (siRNAs), from both bloodstream and procyclic trypanosomes has revealed a new target of the RNAi pathway, namely a family of satellite-like repeats (CIR147) located at chromosomal internal sites. These repeats may function in some aspect of chromosome structure and/or regulation of gene expression. Second, we have identified the RNAi genes in Leishmania braziliensis and in collaboration with Steve Beverley have demonstrated that the RNAi pathway is operational. However, L brasiliensis has some limitations for studying pathogenesis because at present there is no suitable small animal model of the disease. In contrast, Diane McMahon-Pratt at Yale has established a mouse model for L. panamensis, a close relative of L. brasiliensis, which also has the RNAi genes. Thus, our findings open the way to establish tools for applying the wonders of RNAi technology to the study of different aspects of the biology and pathogenesis of Leishmania. Third, we have discovered that a second T. brucei Argonaute-like protein (termed PiwiS) is localized in the mitochondrion and forms foci reminiscent of the antipodal sites where some enzymes for kinetoplast DNA (kDNA) replication localize, implicating PiwiS in some aspect of kDNA biogenesis. In the last few years Argonaute proteins have emerged as central regulators of an outstandingly wide range of phenomena, both at the transcriptional and post-transcriptional levels. Thus, we hypothesize that PiwiS functions in some aspect of RNA silencing in the mitochondrion. It should be noted that this protein is the first example of an Argonaute protein that localizes to an organelle. Our specific aims for the next funding period are: 1. To further mine the biology of trypanosomatids through high-throughput sequencing of small RNAs from T. brucei and from L. braziliensis, and through the functional and structural characterization of the CIR147 satellite-like repeat family. 2. To establish tools for heritable and regulated RNAi in L. panamensis. 3. To establish the function of PiwiS in the T. brucei mitochondrion. Project Narrative: African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Our research has focused towards understanding the mechanism and components of RNA interference to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases.