SMALL RNAS AND THEIR ROLE IN TRYPANOSOME BIOLOGY

小 RNA 及其在锥虫生物学中的作用

• 批准号: 7558950
• 负责人: ELISABETTA ULLU
• 金额: $ 37.24万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7558950
• 关键词: Africa South of the Sahara; African; African Trypanosomiasis; Animal Disease Models; Biogenesis; Biology; Blood Circulation; Chromosome Segregation; Chromosome Structures; Clinical Trials; Collaborations; DNA biosynthesis; Development; Disease; Drug Delivery Systems; Enzymes; Family; Foundations; Funding; Gene Expression Regulation; Genes; Heterochromatin; Kinetoplast DNA; Laboratories; Leishmania; Leishmania braziliensis; Mining; Mitochondria; Organelles; Parasites; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Proteins; Protozoa; RNA Interference; Relative (related person); Research; Role; Site; Small Interfering RNA; Small RNA; Staging; Technology; Trypanosoma; Trypanosoma brucei brucei; human disease; mRNA Transcript Degradation; mouse model; research study; stem; stem cell fate; tool

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.

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，一直致力于研究布氏锥虫的生物学，并为了解锥虫独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，这些靶点可用于对抗寄生虫本身。自 1998 年我们在布氏锥虫中发现 RNAi 途径以来，我们的研究重点是了解 RNAi 的机制和组成部分。这些研究对于最终尝试使用小 RNA 作为对抗锥虫的药物至关重要。这种可能性并非没有根据，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间产生的三个主要发现。首先，对来自血流和原环锥虫的小干扰 RNA (siRNA) 进行测序揭示了 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 (CIR147) 家族。这些重复可能在染色体结构和/或基因表达调节的某些方面发挥作用。其次，我们鉴定了巴西利什曼原虫中的 RNAi 基因，并与 Steve Beverley 合作证明了 RNAi 途径是可操作的。然而，由于目前尚无合适的小动物模型，巴西乳杆菌对于研究发病机制有一定的局限性。相比之下，耶鲁大学的黛安·麦克马洪-普拉特 (Diane McMahon-Pratt) 建立了巴拿马乳杆菌 (L. panamensis) 小鼠模型，它是巴西乳杆菌 (L. brasiliensis) 的近亲，也具有 RNAi 基因。因此，我们的研究结果为建立工具开辟了道路，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。第三，我们发现第二种 T. brucei Argonaute 样蛋白（称为 PiwiS）位于线粒体中，并形成了让人想起一些用于动质体 DNA (kDNA) 复制的酶定位的对足位点的焦点，暗示 PiwiS 在某些方面kDNA 生物发生。在过去的几年中，Argonaute 蛋白已成为转录和转录后水平上一系列极其广泛的现象的中央调节因子。因此，我们假设 PiwiS 在线粒体 RNA 沉默的某些方面发挥作用。值得注意的是，该蛋白是定位于细胞器的 Argonaute 蛋白的第一个例子。我们下一个资助期的具体目标是： 1. 通过对来自 T. brucei 和 L. braziliensis 的小 RNA 进行高通量测序，并通过 CIR147 卫星样的功能和结构表征，进一步挖掘锥虫的生物学特征。重复家庭。 2. 建立巴拿马乳杆菌可遗传和调控的RNAi工具。 3. 建立PiwiS在T. brucei线粒体中的功能。 项目叙述：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，用于消除这种疾病的药物并不理想。我们的研究重点是了解 RNA 干扰的机制和成分，最终尝试使用小 RNA 作为对抗锥虫的药物。这种可能性并非没有根据，因为已经有几项临床试验使用小RNA来治疗某些人类疾病。