TRANSCRIPTIONAL CONTROL OF MITOCHONDRIAL GENE EXPRESSION IN TRYPANOSOMES

锥虫线粒体基因表达的转录控制

• 批准号: 10415106
• 负责人: Ruslan Afasizhev
• 金额: $ 41.05万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-13 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415106
• 关键词: Affect; Affinity Chromatography; Africa South of the Sahara; African; Animals; Area; Biological; Biotinylation; Blood Circulation; Chemicals; Code; Complex; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Developing Countries; Development; Disease; Drug Targeting; Economic Burden; Elements; Eukaryota; Evolution; Excision; Family; Foundations; Gene Expression; Gene Proteins; Genes; Genetic Transcription; Genome; Guide RNA; Histones; Human; In Vitro; Individual; Infection; Insecta; Kinetoplast DNA; Link; Location; Maps; Mass Spectrum Analysis; Messenger RNA; Microscopy; Mitochondria; Mitochondrial DNA; Mitochondrial RNA; Modeling; Molecular; Mutation; Nucleoproteins; Organism; Parasites; Pathogenesis; Polyadenylation; Polymerase; Population; Positioning Attribute; Proliferating; Promoter Regions; Protein Subunits; Proteins; Public Health; RNA; RNA Editing; RNA Processing; RNA chemical synthesis; Recombinants; Research; Resolution; Resources; Ribosomal Proteins; Ribosomal RNA; Role; Site; System; TATA Box; Taxonomy; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Transfer RNA; Trypanosoma; Trypanosoma brucei brucei; Trypanosomiasis; Untranslated RNA; base; crosslink; drug development; experimental study; fascinate; fungus; genome-wide; hemoflagellate; in vivo; innovation; insight; mRNA Precursor; mRNA Transcript Degradation; mitochondrial genome; particle; promoter; protein protein interaction; reconstitution; respiratory; success; targeted treatment; tool; transcription factor; virtual

ABSTRACT Parasitic protist Trypanosoma brucei causes African human and animal trypanosomiasis, a spectrum of diseases affecting the population and economy in sub-Saharan Africa. These digenetic hemoflagellates belong to Kinetoplastea, a taxonomic class distinguished by possession of a kinetoplast. This nucleoprotein body contains mitochondrial DNA (kDNA) of two kinds: ~25 maxicircles (each ~23 kb) encoding rRNAs, ribosomal proteins and subunits of respiratory complexes, and approximately 5000 of ~1 kb minicircles bearing guide RNA genes. Relaxed maxicircles and minicircles are interlinked and packed into a dense disc-shaped network by association with histone-like proteins. Decades of kDNA studies have unraveled fascinating phenomena of general biological significance, such as DNA bending and mRNA editing, and revealed exquisite details of replication and RNA processing. However, the molecular mechanisms of transcription remain virtually unexplored and arguably constitute the most critical gap in understanding mitochondrial gene expression. The historically enduring view of polycistronic RNA synthesis has abridged efforts to investigate transcription's contribution to regulating genome activity. In contrast, this proposal presents evidence that maxicircle and minicircle genes are individually transcribed into 3′ extended precursors. The transcription start site defines pre-mRNA 5′ terminus, which is subsequently converted into monophosphorylated state by a pyrophosphohydrolase complex, termed the PPsome. Most guide RNAs lack PPsome recognition sites and remain triphosphorylated. Furthermore, we establish that antisense transcripts delimit the 3′ boundaries of mature RNAs by blocking 3′-5′ degradation of precursors by the 3′ processome (MPsome). It follows that transcription start sites on sense and antisense strands define 5′ and 3′ mRNA termini, respectively. These findings support a concept of mitochondrial gene- specific transcriptional control with broad implications in parasite development and pathogenesis. We posit that elucidating transcription complex composition, DNA template requirements and functions of specific factors will build a foundation for this nascent research area. We propose to: 1) Characterize RNA polymerase complex from bloodstream and insect parasite forms, and assess transcription factors' contributions to RNA synthesis; 2) Map maxicircle and minicircle promoters; and 3) Reconstitute the active transcription complex.

摘要 寄生原生生物布氏锥虫导致非洲人类和动物锥虫病，一系列疾病 影响到撒哈拉以南非洲的人口和经济。这些二代血鞭毛虫属于 动质体纲，一个以拥有动质体而区别的分类纲。该核蛋白体含有 线粒体DNA（kDNA）有两种：~25个大环（每个~23 kb），编码rRNA、核糖体蛋白和 呼吸复合物的亚基，以及大约5000个~1 kb的携带指导RNA基因的微环。 松弛的大环和小环相互连接，并通过关联包装成致密的盘状网络 组蛋白样蛋白。几十年的kDNA研究揭示了一般生物学的迷人现象， 这些研究揭示了DNA弯曲和mRNA编辑的重要性，并揭示了复制和RNA合成的精致细节。 处理.然而，转录的分子机制仍然几乎未被探索， 构成了理解线粒体基因表达的最关键空白。历史上经久不衰的观点 多顺反子RNA合成的研究简化了研究转录对调节 基因组活性相比之下，这一建议提出的证据表明，大环和小环基因是单独的 转录成3′延伸的前体。转录起始位点定义了前mRNA的5′端， 随后被焦磷酸水解酶复合物转化为单磷酸化状态，称为 PPsome。大多数向导RNA缺乏PPsome识别位点并保持三磷酸化。而且我们 通过阻断3′-5′降解来建立限定成熟RNA 3′边界的反义转录物， 3′加工体（MPsome）。因此，有义和反义上的转录起始位点 5′和3′末端分别由两条链构成。这些发现支持了线粒体基因的概念- 在寄生虫发育和发病机制中具有广泛意义的特异性转录控制。我们将其 阐明转录复合物的组成、DNA模板要求和特定因子的功能， 为这一新兴的研究领域奠定基础。我们的主要工作是：1）RNA聚合酶复合体的表征 从血液和昆虫寄生虫形式，并评估转录因子的贡献RNA合成; 2） 映射大环和小环启动子;和3）重建活性转录复合物。