SMALL RNAS AND THEIR ROLE IN TRYPANOSOME BIOLOGY

描述（由申请人提供）：非洲锥虫在撒哈拉以南非洲的广大地区引起疾病和痛苦，至少可以这么说，可用于消除该疾病的药物不是最理想的。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，并为理解锥虫特有的几种机制做出了贡献。锥虫特异性途径的发现是确定新的潜在药物靶点的第一步，这些靶点可能被用来对抗寄生虫本身。自1998年我们在布鲁氏锥虫中发现RNAi途径以来，我们的研究重点是了解RNAi的机制和成分。这些研究对于最终尝试使用小rna作为抗锥虫的药物至关重要。这种可能性并非毫无根据，因为已经有几个临床试验在使用小rna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间产生的三个主要发现。首先，对来自血液和前环锥虫的小干扰rna （sirna）进行测序，揭示了RNAi途径的新靶点，即位于染色体内部位置的卫星样重复序列（CIR147）家族。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起作用。其次，我们已经确定了巴西利什曼原虫的RNAi基因，并与Steve Beverley合作证明了RNAi途径是可操作的。然而，由于目前还没有合适的小动物模型，对其发病机制的研究存在一定的局限性。相比之下，耶鲁大学的Diane McMahon-Pratt已经建立了一个巴拿马乳杆菌的小鼠模型，它是巴西乳杆菌的近亲，也有RNAi基因。因此，我们的发现为建立将RNAi技术应用于利什曼原虫生物学和发病机制的不同方面的研究工具开辟了道路。第三，我们发现第二种布鲁氏虾argonaute样蛋白（称为PiwiS）定位于线粒体中，并形成与对跖点位点相似的焦点，在对跖点位置，一些酶用于着丝体DNA （kDNA）的复制，这暗示PiwiS在kDNA生物发生的某些方面。在过去的几年里，Argonaute蛋白在转录和转录后水平上都成为了一系列异常广泛的现象的中心调节因子。因此，我们假设PiwiS在线粒体中RNA沉默的某些方面起作用。值得注意的是，这种蛋白是Argonaute蛋白定位于细胞器的第一个例子。我们下一个筹资期的具体目标是：1。通过对布鲁氏T.和巴西L.小rna的高通量测序，以及CIR147卫星样重复序列家族的功能和结构表征，进一步挖掘锥虫的生物学特性。2. 建立巴拿马乳杆菌遗传调控RNAi工具。3. 目的：探讨PiwiS在中华绒螯虾线粒体中的功能。

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.