The Trypanosoma brucei Epitranscriptome

布氏锥虫表观转录组

• 批准号: 9377550
• 负责人: KEVIN MILITELLO
• 金额: $ 45.92万
• 依托单位: COLLEGE AT GENESEO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9377550
• 关键词: Active Sites; African Trypanosomiasis; Bacteria; Biochemical Pathway; Biochemistry; Biological; Biological Assay; Biology; Blood Circulation; Chromatography; Complement; Complex; Computer Simulation; Cytosine; DNA; Data; Dideoxy Chain Termination DNA Sequencing; Disease; Drug Design; Drug Targeting; Drug toxicity; Drug usage; Escherichia coli; Event; Family; Funding; Gene Expression; Genetic Transcription; Genome; Genomics; Growth; Human; In Vitro; Individual; Insecta; Libraries; Life Cycle Stages; Light; Literature; Mammals; Maps; Measures; Messenger RNA; Metabolic Pathway; Metabolism; Methylation; Methyltransferase; New York; Northern Blotting; Nuclear; Organism; Parasites; Pathway interactions; Pattern; Pharmaceutical Preparations; Poly A; Poly(A)+ RNA; Positioning Attribute; Preparation; Process; Proteins; Publishing; RNA; RNA Interference; RNA Processing; RNA methylation; Recombinants; Regulation; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal RNA; Role; S-Adenosylmethionine; Shapes; System; Testing; Therapeutic Intervention; Thin Layer Chromatography; Toxic effect; Translations; Trypanosoma brucei brucei; Universities; Vaccines; Work; base; bisulfite; bisulfite sequencing; cell growth; design; disorder control; experimental study; fighting; improved; in vivo; methyl group; methylome; mutant; new therapeutic target; next generation sequencing; novel therapeutics; sodium bisulfite; teaching laboratory; transcriptome; transcriptome sequencing; undergraduate research

Project Summary Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no efficacious vaccine. Furthermore, drugs used to treat the late stages of the disease are low in number, often toxic, and can be ineffective due to resistance. A thorough understanding of the parasite's biochemistry and its complex life cycle involving insects and mammals will potentially reveal novel targets for therapeutic intervention. Our recent studies indicate that different types of T. brucei RNAs contain the modified base 5-methylcytosine (5-MeC). Although there is a reasonable amount of literature on the role of 5-MeC in DNA molecules, there is little information regarding the role of 5-MeC in RNA molecules in all organisms including T. brucei. In addition, we have identified a family of putative cytosine RNA methyltransferases in T. brucei called TbCRMTs. Interestingly, two of the RNA methyltransferases, TbCRMT4 and TbCRMT5, are required for maximal parasite growth. Overall, our data leads to our hypothesis that RNA methylation and RNA methyltransferases are biologically significant in T. brucei, and RNA methyltransferases could represent drugs targets. This hypothesis will be directly tested in our proposed project. First, the entire 5-MeC methylome of RNA molecules in procyclic form and bloodstream form T. brucei parasites will be elucidated using RNA bisulfite sequencing (RBS-seq). We already have a small RNA methylome from our previous studies and thus we will focus on poly(A)+ RNA (which includes mRNA) and total RNA (which includes ribosomal RNA). Next, the role of the two critical TbCRMTs (4 and 5) in the described RNA methylation events will be evaluated. RBS-seq will be performed on RNA isolated from TbCRMT RNAi lines, and methylation levels will be compared to lines without an activated RNAi system. The effect of TbCRMTs on RNA levels and RNA processing will be determined, which will indicate how TbCRMTs and RNA methylation shape the transcriptome. In addition, the role of the two TbCRMTs in bloodstream form parasite growth will be evaluated, as current antitrypanosomal drugs target bloodstream form parasites. Finally, the two TbCRMTs will be expressed in bacteria, and recombinant TbCRMTs will be evaluated for in vitro cytosine RNA methyltransferase activity. Our studies will potentially improve control of HAT, as they will describe a new RNA metabolic pathway in T. brucei that is essential for parasite growth and survival.

项目概要 布氏锥虫是导致人类非洲锥虫病 (HAT) 的原生动物寄生虫， 又称非洲昏睡病。为此迫切需要新的控制策略 疾病，因为没有有效的疫苗。此外，用于治疗疾病晚期的药物是 数量少，通常有毒，并且由于耐药性而无效。彻底了解 寄生虫的生物化学及其涉及昆虫和哺乳动物的复杂生命周期将有可能揭示新的 治疗干预的目标。我们最近的研究表明，不同类型的 T. brucei RNA 含有 修饰的碱基 5-甲基胞嘧啶 (5-MeC)。尽管有相当数量的文献阐述了 DNA 分子中含有 5-MeC，但有关 5-MeC 在 RNA 分子中的作用的信息很少。 生物体，包括 T. brucei。此外，我们还鉴定了一个假定的胞嘧啶 RNA 家族 T. brucei 中的甲基转移酶称为 TbCRMT。有趣的是，两种 RNA 甲基转移酶 TbCRMT4 和 TbCRMT5，是寄生虫最大生长所必需的。总的来说，我们的数据得出了我们的假设：RNA 甲基化和 RNA 甲基转移酶在 T. brucei 中具有生物学意义，并且 RNA 甲基转移酶 可以代表药物靶标。这个假设将在我们提出的项目中直接得到检验。首先，整个 原环形式和血流形式 T. brucei 寄生虫的 RNA 分子的 5-MeC 甲基化组将被 使用 RNA 亚硫酸氢盐测序 (RBS-seq) 进行阐明。我们已经从我们的研究中获得了一个小RNA甲基化组 之前的研究，因此我们将重点关注 Poly(A)+ RNA（包括 mRNA）和总 RNA（ 包括核糖体RNA）。接下来，两个关键的 TbCRMT（4 和 5）在所述 RNA 中的作用 将评估甲基化事件。 RBS-seq 将对从 TbCRMT RNAi 系中分离的 RNA 进行， 和甲基化水平将与没有激活RNAi系统的品系进行比较。 TbCRMT 对的影响 RNA 水平和 RNA 加工将被确定，这将表明 TbCRMT 和 RNA 甲基化如何 塑造转录组。此外，两个 TbCRMT 在血流形式寄生虫生长中的作用将是 评估，因为当前的抗锥虫药物针对的是血液中的寄生虫。最后，两个 TbCRMT 将在细菌中表达，重组 TbCRMT 将在体外评估胞嘧啶 RNA 甲基转移酶活性。我们的研究将有可能改善 HAT 的控制，因为它们将描述一种新的 RNA 布氏锥虫的代谢途径对于寄生虫的生长和生存至关重要。