RNA CAPPING IN TRYPANOSOMES

锥虫中的 RNA 加帽

• 批准号: 7805454
• 负责人: CHRISTIAN TSCHUDI
• 金额: $ 40.96万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7805454
• 关键词: Adverse effects; African; African Trypanosomiasis; Binding; Biogenesis; Biology; Cattle; Cell Death; Cell physiology; Chagas Disease; Code; Communicable Diseases; Complex; Data; Disease; Domestic Pig; Drug resistance; Enzymes; Family member; Funding; Genes; Genetic; Genome; Goals; Guanine; Hand; Human; Human Bites; Individual; Infection; Laboratories; Leishmaniasis; Life; Life Cycle Stages; Light; Livestock; Messenger RNA; Metabolism; Metals; Methyltransferase; Mini-Exons; Modification; N-terminal; Nuclear; Nuclear Protein; Nuclear Proteins; Organism; Parasite Control; Parasites; Pathway interactions; Pharmaceutical Preparations; Polyadenylation; Polymerase; Process; Proteins; Protozoa; Public Health; RNA; RNA Caps; RNA Polymerase II; RNA Processing; RNA triphosphatase; Resistance; Small Nuclear RNA; South America; Specificity; Spliced Leader RNA; Spliced Leader Sequences; Staging; Structure; Therapeutic; Toxic effect; Trans-Splicing; Transcript; Treatment Cost; Trypanosoma; Trypanosoma brucei brucei; Trypanosomatina; Tsetse Flies; Vaccines; Work; World Health Organization; base; burden of illness; disability-adjusted life years; guanylyltransferase; in vivo; interest; mRNA Precursor; nagana; novel; prevent; protein protein interaction; public health relevance; trait; wasting

DESCRIPTION (provided by applicant): This application focuses on African trypanosomes of the Trypanosoma brucei subspecies, the causative agents of human African trypanosomiasis, a fatal disease unless treated, with a calculated disease burden of about two million Disability Adjusted Life Years. There are no vaccines and the few available therapeutic drugs have serious side effects and decreasing efficacy in light of the emergence of drug-resistant trypanosomes. Our interest in preventing and curing parasite infections is focused on understanding and ultimately exploiting genetic mechanisms that are essential for all stages of the parasite life cycle, but are either absent or fundamentally different in the human host. One possible therapeutic approach is suggested by the observation that trypanosomatids have strikingly unusual mRNA biosynthetic pathways. A key molecule in this process is the RNA polymerase (pol) II-transcribed spliced leader (SL) RNA, because its m7G-capped 5' terminal sequence is trans-spliced onto the 5' end of each mRNA. Furthermore, a subset of small nuclear RNAs involved in RNA processing, including U1, U2, U3 and U4, are also capped, but they are unusual in that the corresponding genes are transcribed by pol III. In the last funding period our work has led to the identification of two pathways that are essential for mRNA metabolism and viability in T. brucei. First, m7G capping of the SL RNA is carried out by a dedicated capping enzyme, TbCGM1, whose depletion leads to cell death. Second, the hypermodified cap 4 structure of mature SL RNA is specifically recognized by a nuclear cap-binding complex, which is unique in the eukaryotic kingdom and whose depletion is also incompatible with life. Furthermore, we have made the first step towards defining the cellular function of the previously identified capping enzyme TbCE1, namely m7G capping of some of the U-snRNAs; this is a distinctive trait not found in any other eukaryotic organism. Most interestingly, the N-terminal domain of TbCE1 harbors a metal-independent RNA triphosphatase activity with no structural or functional similarities to the previously characterized T. brucei RNA triphosphatase TbCet1 and both triphosphatases are fundamentally different from the triphosphatase present in the human host. The long- term goal of this proposal is to understand the parasite-specific biology and mechanisms of RNA capping in T. brucei and to identify essential factors, factor domains, or protein-protein interactions which might be exploited for parasite control. In the next funding period we plan to: 1. Carry out a functional analysis of the SL RNA m7G capping machinery and the novel metal-independent triphosphatase domain of TbCE1. 2. Identify determinants that enable selective capping of the pol II-transcribed SL RNA and a subset of pol III transcripts. 3. Further characterize the nuclear cap binding complex to elucidate the basis for its specificity for the cap 4 structure and define its in vivo function more precisely. PUBLIC HEALTH RELEVANCE: Parasitic protozoa are a major cause of global infectious diseases and thus, represent one of the most serious threats to public health. Among these are African trypanosomes, the causative agents of African trypanosomiasis or sleeping sickness in humans and a wasting and fatal disease (Nagana) in cattle, domestic pigs and other farm animals causing a profound effect on the economy of much of the continent. Unless treated, African sleeping sickness is always fatal; no vaccine has been approved and there is a very limited arsenal of drugs with generally severe shortcomings, such as high toxicity and emerging resistance.

描述（由申请人提供）：本申请关注布氏锥虫亚种的非洲锥虫，其是人类非洲锥虫病的病原体，非洲锥虫病是一种致命疾病，除非接受治疗，计算的疾病负担约为200万残疾调整生命年。由于抗药性锥虫的出现，没有疫苗，现有的少数治疗药物具有严重的副作用，疗效也在下降。我们对预防和治疗寄生虫感染的兴趣集中在理解和最终利用对寄生虫生命周期的所有阶段都至关重要的遗传机制，但在人类宿主中要么不存在，要么根本不同。一种可能的治疗方法是通过观察锥虫具有惊人的不寻常的mRNA生物合成途径而提出的。这个过程中的一个关键分子是RNA聚合酶（pol）II转录的剪接前导（SL）RNA，因为它的m7 G加帽的5'末端序列被反式剪接到每个mRNA的5'末端。此外，参与RNA加工的小核RNA的子集，包括U1，U2，U3和U4，也被加帽，但它们是不寻常的，因为相应的基因由pol III转录。在上一个资助期间，我们的工作已经确定了两个途径，这两个途径对T.布鲁塞。首先，SL RNA的m7 G加帽通过专用加帽酶TbCGM 1进行，其耗尽导致细胞死亡。第二，成熟SL RNA的高度修饰的帽4结构被核帽结合复合物特异性识别，这在真核生物界中是独特的，并且其耗尽也与生命不相容。此外，我们已经朝着定义先前鉴定的加帽酶TbCE 1的细胞功能迈出了第一步，即一些U-snRNA的m7 G加帽;这是在任何其他真核生物中都没有发现的独特特征。最有趣的是，TbCE 1的N-末端结构域具有不依赖金属的RNA三磷酸酶活性，与先前表征的T.布鲁氏菌RNA三磷酸酶TbCet 1和这两种三磷酸酶与人类宿主中存在的三磷酸酶根本不同。这项计划的长期目标是了解寄生虫特异性生物学和T。布氏杆菌，并确定必要的因素，因素域，或蛋白质-蛋白质相互作用，可能利用寄生虫控制。在下一个融资周期，我们计划：1。对SL RNA m7 G加帽机制和TbCE 1的新型金属非依赖性三磷酸酶结构域进行功能分析。2.鉴定能够选择性加帽pol II转录的SL RNA和pol III转录物子集的决定簇。3.进一步表征核帽结合复合物，以阐明其对帽4结构特异性的基础，并更精确地定义其体内功能。公共卫生相关性：寄生原生动物是全球传染病的主要原因，因此是对公共卫生的最严重威胁之一。其中包括非洲锥虫，它是非洲锥虫病或人类昏睡病的病原体，也是牛、家猪和其他农场动物的一种消耗性和致命性疾病（Nagana），对非洲大陆大部分地区的经济造成深远影响。除非得到治疗，非洲昏睡病总是致命的;没有任何疫苗得到批准，而且药物库非常有限，普遍存在严重缺陷，如毒性高和正在出现耐药性。