RNA CAPPING IN TRYPANOSOMES

锥虫中的 RNA 加帽

• 批准号: 8056644
• 负责人: CHRISTIAN TSCHUDI
• 金额: $ 40.55万
• 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/8056644
• 关键词: Adverse effects; African; African Trypanosomiasis; Binding; Biogenesis; Biology; Cattle; Cell Death; Cell physiology; Central America; Chagas Disease; Code; Communicable Diseases; Complex; Data; Disease; Domestic Pig; Drug resistance; Enzymes; Family member; Funding; Genes; Genetic; Genome; Goals; Guanine; Hand; Health; 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; 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; 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）：本申请的重点是布鲁氏锥虫亚种的非洲锥虫，人类非洲锥虫病的病原体，除非治疗，否则是一种致命的疾病，计算的疾病负担约为200万残疾调整生命年。由于出现了耐药锥虫，目前没有疫苗，可用的少数治疗药物有严重的副作用，而且疗效正在下降。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缺失，要么根本不同。一种可能的治疗方法是通过观察到锥虫具有非常不寻常的mRNA生物合成途径。这个过程中的一个关键分子是RNA聚合酶（pol） ii转录的剪接前体（SL） RNA，因为它的m7g -cap 5‘端序列被反接到每个mRNA的5’端。此外，参与RNA加工的一组小核RNA，包括U1， U2， U3和U4，也被盖住，但它们的不寻常之处在于相应的基因是由pol III转录的。在上一个资助期，我们的工作导致了布鲁氏t mRNA代谢和生存能力必不可少的两个途径的鉴定。首先，SL RNA的m7G盖帽是由一种专用的盖帽酶TbCGM1完成的，TbCGM1的耗尽会导致细胞死亡。其次，成熟SL RNA的超修饰cap 4结构被核cap结合复合体特异性识别，这在真核生物王国中是独一无二的，而且它的耗尽也与生命不相容。此外，我们已经在确定先前鉴定的盖帽酶TbCE1的细胞功能方面迈出了第一步，即一些u - snrna的m7G盖帽；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，TbCE1的n端结构域含有一种不依赖金属的RNA三磷酸酶活性，与先前表征的布鲁氏体RNA三磷酸酶TbCE1没有结构或功能上的相似性，而且这两种三磷酸酶与人类宿主中的三磷酸酶根本不同。本研究的长期目标是了解布鲁氏绦虫的特异性生物学和RNA盖帽机制，并确定可能用于寄生虫控制的必要因子、因子结构域或蛋白-蛋白相互作用。在下一个融资期，我们计划：对SL RNA m7G盖帽机制和TbCE1的新型金属非依赖性三磷酸酶结构域进行功能分析。2. 确定能够选择性封顶pol ii转录的SL RNA和pol III转录物子集的决定因素。3. 进一步表征核帽结合复合物，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，它是人类非洲锥虫病或昏睡病的病原体，以及牛、家猪和其他农场动物的消耗和致命疾病（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；目前尚未批准任何疫苗，而且药物库非常有限，通常存在严重缺陷，例如高毒性和新出现的耐药性。