RNAi in DNA Sequence Elimination in Tetrahymena

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（siRNA）。虽然这种机制可能起源于对入侵的RNA病毒和转座因子的防御，但类似的机制用于各种重要的细胞过程中，以产生参与异常转录物降解、特异性mRNA利用的发育调节、异染色质沉默、着丝粒功能和通过抑制转座因子活性来维持基因组稳定性的特异性细胞内RNA。四膜虫的基因组重排涉及通过生殖细胞微核发育成体细胞大核过程中发生的可重复间质缺失消除约15%的基因组。这一过程由小RNA控制，小RNA靶向序列，使用类似于其他真核生物中异染色质形成的RNAi相关机制消除。这些小RNA的特性解释了特定序列如何被靶向消除，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。该系统提供了直接改变DNA序列组织的RNAi介导过程的第一个演示。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，也特异性地发生在经历消除的序列上，并且依赖于小RNA。这些观察结果有助于染色质生物学的一个新范式：在真核生物中通过RNAi相关机制靶向异染色质形成。消除过程顺序发生，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和/或生殖细胞核，蛋白质标签，串联亲和纯化和几乎完整的四膜虫大核基因组序列中的一个或两个轻松的基因替换，我们建议的小RNA介导的基因组重组的分子机制的特点。这些研究应该揭示其他RNAi样过程，这是重要的正常发展和维持基因组稳定性的多细胞生物。

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.