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.

项目摘要 布鲁氏锥虫是负责人类非洲锥虫病（HAT）的原生动物寄生虫， 否则被称为非洲睡眠疾病。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，用于治疗该疾病晚期的药物是 数量较低，通常有毒，并且由于耐药性可能是无效的。对 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可能会揭示出新颖的 治疗干预的目标。我们最近的研究表明，不同类型的t. brucei rNA包含 改良的基碱5-甲基胞嘧啶（5-MEC）。尽管关于角色的作用有合理的文献 5-MEC在DNA分子中，关于5-MEC在RNA分子中的作用的信息很少 包括布鲁氏菌在内的生物。此外，我们已经确定了一个假定的胞嘧啶RNA家族 在布鲁氏菌中的甲基转移酶称为TBCRMT。有趣的是，两个RNA甲基转移酶TBCRMT4 最大寄生虫生长所必需的TBCRMT5。总体而言，我们的数据导致我们的假设是RNA 甲基化和RNA甲基转移酶在Brucei和RNA甲基转移酶中具有生物学意义 可以代表药物靶标。该假设将在我们提出的项目中直接检验。首先，整个 5-MEC的RNA分子的procyclic形式和血液形式T. Brucei寄生虫将是 使用RNA Bisulfite测序（RBS-SEQ）阐明。我们已经有一个小的RNA甲基团 先前的研究，因此我们将重点介绍poly（a）+ RNA（包括mRNA）和总RNA（它们 包括核糖体RNA）。接下来，两个临界TBCRMT（4和5）在所述RNA中的作用 将评估甲基化事件。 RBS-seq将在从TBCRMT RNAi线分离的RNA上进行， 并将甲基化水平与没有激活的RNAi系统的线进行比较。 TBCRMT对 将确定RNA水平和RNA处理，这将指示TBCRMT和RNA甲基化如何 塑造转录组。此外，两个TBCRMT在血液形式的寄生虫生长中的作用将是 评估当前的抗丙糖体药物靶向血液形成寄生虫。最后，两个tbcrmt 将在细菌中表达，重组TBCRMT将用于体外胞嘧啶RNA 甲基转移酶活性。我们的研究将有可能改善对帽子的控制，因为他们将描述一个新的RNA T. Brucei的代谢途径对于寄生虫生长和生存至关重要。