RNAi in DNA Sequence Elimination in Tetrahymena

描述(申请人提供)：动物、植物、真菌和原生生物的细胞通过一种保守的机制(RNAi机制)对细胞内双链RNA(DsRNA)的存在做出反应，该机制将dsRNA切割成小干扰RNA(SiRNAs)。虽然这种机制可能起源于对入侵的RNA病毒和转座子的防御，但类似的机制也用于各种重要的细胞过程中，产生特定的细胞内RNA，涉及异常转录的降解、特定mRNAs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子的活性来维持基因组的稳定性。四膜虫的基因组重排涉及到在体细胞大核从胚系微核发育过程中发生的可重复的间质缺失，从而消除约15%的基因组。这一过程由小RNA控制，这些小RNA利用与RNAi相关的机制，利用类似于其他真核生物中异染色质形成的RNAi相关机制，针对要消除的序列进行消除。这些小RNA的性质解释了特定序列如何被靶向消除，以及亲本大核的初级序列如何在表观遗传上控制新大核中的基因组重排。该系统首次展示了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上的甲基化和染色域蛋白的积累，这是异染色质的标志，也专门发生在经历消除的序列上，并依赖于小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相关机制来靶向异染色质的形成。消除过程是按顺序进行的，并且可以在大量细胞中高度同步。利用最近发展起来的简便的体细胞核和/或生殖核中的基因替换、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的方法，我们建议表征小RNA介导的基因组重组的分子机制。这些研究应该有助于揭示其他类似RNAi的过程，这些过程对于多细胞生物体的正常发育和维持基因组稳定性非常重要。

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.