RNAi in DNA Sequence Elimination in Tetrahymena

四膜虫 DNA 序列消除中的 RNAi

• 批准号: 7614257
• 负责人: Martin A Gorovsky
• 金额: $ 36.42万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614257
• 关键词: Affinity Chromatography; Animals; Biology; Boxing; Cell Count; Cell Nucleus; Cell physiology; Cells; Centromere; Chromatin; Cleaved cell; Complex; DNA Sequence; DNA Sequence Rearrangement; DNA Transposable Elements; Development; Double-Stranded RNA; Eukaryota; Genes; Genome; Genome Stability; Germline Micronucleus; Goals; Heterochromatin; Histone H3; Invaded; Light; Lysine; Macronucleus; Maintenance; Mammals; Mediating; Methods; Methylation; Methyltransferase; Molecular; Organism; Play; Process; Property; Protein Family; Proteins; RNA Interference; RNA Viruses; RNA-Directed RNA Polymerase; Regulation; Ribonuclease III; Role; Small Interfering RNA; Small RNA; Staging; System; Testing; Tetrahymena; Tetrahymena thermophila; Transcript; gene replacement; genome sequencing; helicase; human DICER1 protein; insight; interstitial; plant fungi; protein function

DESCRIPTION (provided by applicant): Cells of animals, plants, fungi and protists respond to the presence of intracellular double stranded RNA (dsRNA) by a conserved mechanism (the RNAi machinery), which cleaves dsRNA to small interfering RNAs (siRNAs). While this mechanism likely originated as a defense against invading RNA viruses and transposable elements, similar machineries are used in a variety of important cellular processes to produce specific intracellular RNAs involved in degradation of aberrant transcripts, developmental regulation of utilization of specific mRNAs, heterochromatic silencing, centromere function and maintenance of genome stability by suppression of activity of transposable elements. Genome rearrangement in Tetrahymena involves elimination of about 15% of the genome by reproducible interstitial deletions that occur during development of the somatic macronucleus from the germline micronucleus. This process is controlled by small RNAs that target the sequences for elimination using an RNAi-related mechanism that resembles heterochromatin formation in other eukaryotes. The properties of these small RNAs explain how specific sequences are targeted for elimination and how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus. This system provides the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These observations contributed to a new paradigm of chromatin biology: targeting of heterochromatin formation by RNAi-related mechanisms in eukaryotes. The elimination process occurs sequentially and can be highly synchronized in a large number of cells. Utilizing recently developed methods for facile gene replacement in either or both the somatic and/or germline nucleus, protein tagging, tandem affinitiy purification and the nearly complete Tetrahymena macronuclear genome sequence, we propose to characterize the molecular mechanism of small RNA-mediated genome reorganization. These studies should shed light on other RNAi- like processes that are important for normal development and maintenance of genome stability in multicellular organisms.

描述（由申请人提供）：动物、植物、真菌和原生生物的细胞通过保守机制（RNAi 机制）对细胞内双链 RNA (dsRNA) 的存在作出反应，该机制将 dsRNA 裂解为小干扰 RNA (siRNA)。虽然这种机制可能起源于防御入侵的RNA病毒和转座元件，但类似的机制被用于各种重要的细胞过程中，以产生特定的细胞内RNA，这些RNA参与异常转录本的降解、特定mRNA的利用的发育调节、异染色质沉默、着丝粒功能以及通过抑制转座元件的活性来维持基因组稳定性。四膜虫的基因组重排涉及通过可重复的间质缺失消除约 15% 的基因组，这种缺失是在种系微核发育成体细胞大核的过程中发生的。这一过程由小 RNA 控制，这些小 RNA 使用类似于其他真核生物中异染色质形成的 RNAi 相关机制来靶向消除序列。这些小RNA的特性解释了如何靶向消除特定序列以及亲本大核的一级序列如何表观遗传地控制新大核中的基因组重排。该系统首次演示了 RNAi 介导的直接改变 DNA 序列组织的过程。组蛋白 H3 在赖氨酸 9 上的甲基化和染色质结构域蛋白的积累（异染色质的标志）也专门发生在正在消除的序列上，并且依赖于小 RNA。这些观察结果促成了染色质生物学的新范式：通过真核生物中 RNAi 相关机制靶向异染色质形成。消除过程是连续发生的，并且可以在大量细胞中高度同步。利用最近开发的方法在体细胞核和/或种系核中进行简单的基因替换、蛋白质标记、串联亲和纯化和几乎完整的四膜虫大核基因组序列，我们建议表征小RNA介导的基因组重组的分子机制。这些研究应该揭示其他类似 RNAi 的过程，这些过程对于多细胞生物体的正常发育和基因组稳定性的维持很重要。