“十二五” 国家重点图书出版规划项目

生命科学前沿

Celluar RNA Interference Mechanisms

细胞 RNA 干扰机制

(导读版)

〔德〕 D． 格林　 编著

李慎涛　 导读

北　 京

图字: 01-2012-5511 号内 容 简 介

本书是 Elsevier 学术出版社 Progress in Molecular Biology and Translational Science 丛书的第 102集, 为 “细胞 RNA 干扰机制” 专题。 详细介绍了 RNAi 的基本理论和实验技术, 内容比较前沿和实用, 与国内研究热点相吻合, 对国内 RNAi 研究具有较好的指导意义。 适合于从事分子生物学和相关领域的研究人员、 生物学和医学专业研究生、 药物设计与开发研究人员使用。

This is an annotated version ofCelluar RNA Interference Mechanisms

Edited by Dirk GrimmCopyright© 2012 Elsevier Inc．

ISBN: 978-0-12-415795-8

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导　 　 读

20 世纪末, RNA 干扰 (RNA interference, RNAi) 现象被发现。 2001 年, 科

学家应用 RNAi 技术成功地介导了哺乳动物培养细胞的特异性基因沉默, 被 《科学》 杂志评为当年的十大科学进展之一, 并名列 2002 年十大科学进展之首。2006 年, 美国科学家 Andrew Z． Fire 和 Craig C． Mello 因在 1998 年阐明了 RNA 干

扰现象而荣获诺贝尔医学或生理学奖。 自此以后, RNAi 的研究工作被推向高潮。RNA 干扰是指内源性或外源性双链 RNA (double-stranded RNA, dsRNA) 导

致细胞内同源 mRNA 发生特异性降解的过程。 RNA 干扰现象普遍存在于真菌、果蝇、 线虫、 涡虫、 植物和动物等大多数真核生物中, 它可以减弱或阻断生物体

内特定基因的表达, 从而使细胞表现出特定基因缺失的表型, 是一种在进化上高

度保守的调节机制。 自发现 RNA 干扰现象以来, RNA 干扰技术迅速发展成为一

种强大的基因沉默工具, 是当今生物学研究的一大热点, 到目前为止, 对 RNA干扰作用机制和作用特点的研究已取得了很大的进展。

RNA 干扰在基因沉默方面具有高效性、 简单性和特异性, 是研究基因功能

的重要工具, 可对基因的表达和调控进行分析, 并在多个领域具有很好的应用价

值。 例如, 在植物研究方面, 可利用 RNA 干扰技术改良作物品种和培育抗病虫

害的品种等。 在基因药物研发方面, RNA 干扰是确认药物靶标的一种重要工具。同时, 那些在靶标实验中被证明有效的 siRNA / shRNA 本身还可以被进一步开发

成为 RNA 干扰药物。 在疾病治疗方面, 双链小分子 RNA 或 siRNA 已被用于几种

疾病 (如老年视黄斑退化、 肌肉萎缩性侧索硬化症、 类风湿性关节炎、 肥胖症

等) 治疗的临床测试, 并在帕金森病等神经系统疾病开始尝试 RNA 干扰疗法。在抗病毒治疗方面, 对乙型肝炎 (HBV) 的治疗也进行了尝试, 其原因是 RNA干扰直接针对 mRNA, 能够有效地抑制乙肝病毒抗原的生成, 消除免疫系统对肝

脏的损伤, 从而限制甚至阻止乙肝病毒携带者向慢性乙肝、 肝硬化和肝癌发展。尽管 RNA 干扰技术目前存在一些给药途径和安全性的问题, 但这一项新技术为

治疗 HBV 慢性感染开拓了一个全新的方向, 而且 RNA 干扰技术不存在耐药问

题。 相信在不久的将来我们能将 RNA 干扰技术应用于抑制体内 HBV 病毒的复

制, 甚至达到清除病毒的目的。 此外, RNA 干扰在对流感病毒 A、 人免疫缺陷病

毒 (HIV)、 人乳头瘤病毒 (HPV) 的研究中也有应用并取得了较好的结果。 在

肿瘤治疗方面, 目前还处于摸索和积累阶段, 已有相关的报道。 例如, DeSchrijver 等 (2003) 用 RNA 干扰技术降低了人类上皮癌中高表达脂肪酸酶

(FASE) 的表达, 并使前列腺癌细胞发生凋亡。 有学者用 RNA 干扰成功地抑制

了表皮生长因子、 血管内皮生长因子和 BCL2 的表达, 并用 RNA 干扰对白血病、结肠癌等的治疗进行了研究, 取得了一定的效果。 随着研究的不断深入, RNA干扰将在各研究领域发挥越来越重要的作用。

我国 RNA 干扰研究起步于 2001 年, 总体说来, 国内在该领域的科研工作与

国外差距不大, 许多研究单位也都具备了开展 RNAi 研究的条件, 并掌握了相关

的技术。 但在应用研究方面, 我国与国外差距较大, 缺乏一些原创性的科研成

果。 在国家知识产权局网站可检索到的有关 RNAi 的专利仅有 302 项, 其中还有

很多是国外公司在我国申请的专利。本书是 Elsevier 学术出版社 Progress in Molecular Biology and Translational

Science 丛书的第 102 集, 该丛书每集都集中在一个专题, 例如 “蛋白质折叠的分

子生物学”、 “基因与肥胖”、 “人类疾病的动物模型”、 “DNA 修复的机制” 和

“干细胞遗传学” 等, 由本领域的著名专家撰写, 独成体系。 本集为 “细胞 RNA干扰机制” 专题, 由 Dirk Grimm 编纂, 全书共分 6 章, 主要内容如下。

第一章: 由美国加利福尼亚拉霍拉斯克里普斯研究所分子与实验医学系的

Stuart Knowling 和 Kevin V． Morris 撰写。 该章对本领域一个最新发现进行了综述,即小 RNA 不仅在转录后水平调节基因的表达, 而且还在转录过程中发挥调节作

用。 而且小 RNA 既能够抑制基因的表达, 也能够活化基因的表达, 虽然目前还

不清楚其机制, 但这必将成为今后的研究方向。 作者也希望将这些新发现转化为

未来生物医学的新概念, 使我们能够在另一个重要层次上控制人类基因的表达。第二章: 由南非约翰内斯堡威特沃特斯兰大学分子医学与血液病学系抗病毒

基因治疗研究组的 Victoria A． Green 和 Marc S． Weinberg 撰写, 深入探究了人类细

胞中决定表观遗传学 RNAi 调节过程的各种分子机制, 为我们大体理解人类细胞

的功能和药物∕基因治疗展现了 RNAi 的应用价值。第三章: 由德国海德堡大学的 Roberto Fiore、 Sharof Khudayberdiev、 Reuben

Saba 和 Gerhard Schratt 撰写, 介绍了在中枢神经系统中由 miRNA 引发的转录后基

因沉默。 RNAi 如何决定和控制诸如神经元分化、 突触发生和可塑性等中枢细胞

活动? 在本章, 作者进行了初步的介绍, 也对 RNAi 与高级认知功能和神经系统

疾病的病因学之间的关系也进行了初步探讨。第四章: 由英国诺丁汉大学生物分子科学中心药学院的 Ashley P． E． Roberts、

Andrew P． Lewis 和 Catherine L． Jopling 撰写, 首先对病毒的结构与功能进行了概

述, 继而展示了与 miRNA 和人类 RNAi 机器有关的病毒—宿主相互作用的复杂

性, 介绍了病毒感染对细胞 miRNA 的调节以及细胞 miRNA 对病毒的调节作用。详细阐明了人丙型肝炎病毒与一种肝特异性 miRNA 相互作用, 相信读者会对此

感兴趣。

第五章: 由荷兰阿姆斯特丹大学学术医疗中心感染与免疫中心医学微生物学

系实验病毒学实验室的 Julia J． M． Eekels 和 Ben Berkhout 撰写, 重点介绍了怎样

用细胞 RNAi 机制对抗病毒入侵, 研制基于 RNAi 的临床治疗方法, 以对抗 AIDS病毒并将其消灭, 同时也使其没有机会通过自然突变来逃避治疗。 并介绍了针对

病毒和细胞多个必需基因的组合 RNAi 打靶技术, 以清除 HIV 感染者体内的

病毒。第六章: 由德国海德堡大学 Cluster of Excellence CellNetworks 传染病与病毒

学系的 Stefan Mockenhaupt、 Nina Schürmann 和 Dirk Grimm 撰写, 介绍了他们课题

组的工作, 全面回顾了在人类肿瘤和其他致命性疾病中可能失调的细胞 RNAi 机制, 讨论了大量新治疗概念和转化途径, 重申了 RNAi 基础研究和应用研究之间

的紧密关系。 也把重点放在病毒上, 讨论相关的最新观察和得到的深刻教训, 并

以展望的形式结束本书。作者 Dirk Grimm 于 1998 年获得德国海德堡大学的生物学博士学位, 1999 ~

2001 年在德国癌症研究中心从事博士后研究, 2001 ~ 2006 年在斯坦福大学医学

院从事博士后研究, 2006 ~ 2007 年为斯坦福大学医学院的 “Research Associate”,自 2007 年起在德国海德堡大学医院 Cluster of Excellence CellNetworks 传染病与病

毒学系工作, 任 “病毒—宿主相互作用” 研究组组长。 主要研究领域为: 腺相

关病毒 (AAV) 载体的构建及其优化、 人致病性病毒与宿主细胞的天然相互作

用、 细胞 RNA 干扰 (RNAi) 的机制及其在病毒感染和致病过程中的作用、 高通

量 RNAi 筛选方法的设计、 基于 AAV / RNAi 的新治疗方法的研究等。 至今已发表

30 多篇论文, 主要涉及 AAV 病毒载体的研制与应用、 RNAi 的机制和应用等。2006 年以第一作者身份在 《自然》 杂志上发表了题为 Fatality in Mice due to Over-saturation of Cellular microRNA / short Hairpin RNA Pathways 的研究论文, 探讨了小

RNA 临床应用可能存在的副作用, 具有很好的理论意义的应用价值。本书的其他作者也都是在本研究领域卓有成就的学者和企业专家, 他们具有

丰富的理论知识背景和实际应用的经验, 许多章节都是作者自己的研究总结, 所

涉及的内容比较前沿和实用, 代表了当今 RNAi 研究的潮流, 其内容与目前国内

的研究热点相吻合, 对国内 RNAi 研究具有较好的指导意义。本书既注重理论知识, 也有具体的研究方法介绍, 每章后面附有相关的参考

文献, 这些文献涵盖了 RNAi 研究领域的方方面面, 具有很好的代表性。 但由于

本书以专题的形式出版, 各章节之间缺乏连贯性、 理论介绍缺乏系统性, 没有一

定生物学知识背景的读者读起来可能会感到吃力。 为了更好地理解本书的内容,建议读者参阅科学出版社出版的 《RNAi: 基因沉默指南 (影印版)》 和人民卫生

出版社的 《实用 RNAi 技术: 线虫、 果蝇和哺乳动物细胞基因沉默的基本原则与

方法 (翻译版)》, 前者由著名专家撰写, 内容全面、 系统性强、 基本概念清晰;后者除了基本理论外, 还含有大量 RNAi 相关的实验方法, 具有很好的指导作

用。 这两本书与本书结合起来, 可以起到互相补充的作用。 该书适合于从事分子

生物学和相关领域的研究人员、 生物学和医学专业研究生、 药物设计与开发研究

人员使用。

李慎涛

2013 年 6 月 10 日于首都医科大学

前　 　 言

几个世纪以来, 大量的生物学发现使我们对自然有了基本的认识和理解, 并

且使人类疾病的诊断与治疗方法得以改进。 尽管这样, RNA 干扰技术 (RNAi;一种在进化上保守、 特异且功能强大的基因沉默方法) 在哺乳动物细胞中的应用

及其所产生的双重作用是其他研究所不能比拟的。 的确, 对高等生物基因调节巨

大复杂性的认识, 自 RNAi 首次在线虫上报道以来, 在过去的几十年里让我们目

睹了一场不折不扣的革命, 即便如此, 我们可能才刚开始意识到众多相关机制的

内在协调性和复杂多样性。 同时, 我们对人类疾病分子过程的认知正在不断地深

化, 为促进诊断和预后工具的研发, 以及进一步拓展临床干预策略提供了全新而

有益的途径。本书共分 6 章, 分别对健康人和患者 “细胞 RNAi 机制” 的惊人复杂性和多

样性进行了介绍, 希望能够给读者留下这样的印象: 整个领域正在以惊人的速度

前进, 并不断促进生物学与医学的融合, 这是前所未有的。本书第 1 章由 Stuart Knowling 和 Kevin V． Morris 撰写, 对所属领域一个最新

和意想不到的发现进行了综述, 即小 RNA 不仅在转录后水平调节基因的表达

(经典的 RNAi 通路), 同时还在转录过程中发挥调节作用。 此外, 人们才刚刚开

始认识到, 小 RNA 不仅抑制基因表达, 也能够活化基因表达。 尽管目前尚不清

楚其机制, 但在未来的研究中将成为引人关注的研究课题。 按照本书章节和

RNAi 领域文献的惯例, Knowling 和 Morris 在本章的最后提出, 希望这些新发现

能被转化为未来生物医学的新概念, 使人们能在又一个重要层次上控制人类基因

的表达。在先导概述之后, Victoria A． Green 和 Marc S． Weinberg 撰写了第 2 章, 作为

第 1 章的补充, 作者深入探究了人类细胞中支配一些非经典表观遗传学 RNAi 调节过程的大量潜在分子机制。 尽管目前我们还不能完全领会大量基于非编码

RNA 并在基因表达水平上发挥作用的调控网络, 但是 Green 和 Weinberg 已经为

我们展现了这些新发现的潜在应用价值, 使我们大致理解人类细胞的功能, 以及

在未来药物和基因治疗研发方面使用全新的生物医学策略。离开 细 胞 核, 看 一 下 细 胞 质 中 的 基 因 调 节, Roberto Fiore、 Sharof

Khudayberdiev、 Reuben Saba 和 Gerhard Schratt 接下来介绍了当前 RNAi 研究的另

一个热点领域, 即在中枢神经系统中由 miRNA 引发的转录后基因沉默。 此外,RNAi 如何决定和控制诸如神经元分化、 突触发生和可塑性等中枢细胞活动? 他

们进行了初步的介绍, 同时也初步阐述了 RNAi 与高级认知功能和神经系统疾病

病因学之间的关系。 所有这些都是读者渴望得到解答的令人着迷的问题, 相信在

未来的岁月里, 读者的答案将会不断地革新乃至颠覆目前我们对健康人和患者神

经系统细胞 RNAi 机制的认识。作为对上述三篇人类细胞内在 RNAi 过程的补充, 后续两章进入了令人关注

的致病性病毒领域, 从分子生物学和转化科学的观点分析了病毒与细胞 RNAi 机制的关系。 首先, Ashley P． E． Roberts、 Andrew P． Lewis 和 Catherine L． Jopling 将

对病毒的结构与功能进行广泛而深入的概述, 继而展示与 miRNA 和人类 RNAi 机器有关的病毒—宿主相互作用不可思议的复杂性。 作者随后将详细讨论一个特殊

的案例, 即人丙型肝炎病毒 (HCV) 与一种肝特异性 miRNA 的相互作用, 这种

相互作用非同寻常, 在过去的几年里, 一直使 RNAi 和病毒研究人员充满惊奇,也希望能够激发本章读者的兴趣。

接续病毒如何利用和调节人类 RNAi 机器的上述内容, Julia J． M． Eekels 和

Ben Berkhout 转而着重介绍了人类怎样利用这种细胞机制来对抗入侵者, 研制基

于 RNAi 的临床手段, 不仅用于对抗 AIDS 病毒 HIV-1 并将其消灭, 同时也使病

毒没有任何机会通过自然突变来逃避治疗。 为此, 作者首先全面回顾了大量的策

略和工具, 多为最近被论证、 建立和验证的。 然后介绍了针对病毒和细胞多个必

需基因的组合 RNAi 打靶技术, 被认为是最新和最有前途的治疗方法, 以对付感

染者体内的 HIV 病原并将其永久清除。本书最后一章介绍了本课题组的工作, 全面回顾了在人类肿瘤和其他致命疾

病中可能失调的细胞 RNAi 机制, 同时讨论了新文献中不断涌现的新治疗概念和

转化途径, 重申了 RNAi 基础研究和应用研究之间非常紧密的关系。 为衔接前面

的章节, 我们也把重点放在病毒上, 这不仅因为病毒是用分子干预方法扰乱、 劫

持和重编程细胞过程的最佳例子, 还因为我们正努力将病毒改造成表达 RNAi 的工具来治疗人类疾病, 只是这些病毒偶尔会变成我们最可怕的敌人, 无意间摧毁

人类的 RNAi 机器。 Stefan Mockenhaupt、 Nina Schürmann 和 Dirk Grimm 首先将讨

论有关的最新观察和得到的深刻教训, 最后会以展望的形式结束本章和全书: 要

进一步理解和利用细胞 RNAi 机制, 并确保整个领域在未来的岁月里更加繁荣,什么将是下一步重要的举措?

Dirk Grimm

目　 　 录

导读

前言

人类细胞中非编码 RNA 对基因表达的表观遗传学调节

　 Stuart Knowling 和 Kevin V． MorrisI． 　 非编码 RNA 简介 1…………………………………………………………

II． 表观遗传学与非编码 RNA 3………………………………………………

III． 基因间长非编码 RNA 与基因转录的表观遗传学控制 4…………………

参考文献 8…………………………………………………………………………

哺乳动物小 RNA 诱导的转录基因调节: 机制、 治疗应用和在基因组的范畴

　 Victoria A． Green 和 Marc S． WeinbergI． 简介 12………………………………………………………………………

II． 哺乳动物小 RNA 生物发生通路 13…………………………………………

III． 小 RNA 诱导的转录调节机制 16……………………………………………

IV． 转录水平基因沉默的治疗应用 25…………………………………………

V． 转录水平基因沉默治疗剂的范围 30………………………………………

VI． 结论 37………………………………………………………………………

参考文献 38………………………………………………………………………

microRNA 在神经系统中的功能

　 Roberto Fiore、 Sharof Khudayberdiev、 Reuben Saba 和 Gerhard SchrattI． 简介 48………………………………………………………………………

II． 神经分化中的 miRNA 49……………………………………………………

III． 有丝分裂后神经元中的 miRNA 60…………………………………………

IV． miRNA 在神经系统疾病中的作用 69………………………………………

参考文献 91………………………………………………………………………

microRNA 在病毒感染中的作用

　 Ashley P． E． Roberts、 Andrew P． Lewis 和 Catherine L． JoplingI． 简介 102………………………………………………………………………

II． miRNA 的调节及其与疾病的关系 108……………………………………

III． 病毒感染简介 111……………………………………………………………

IV． 病毒 miRNA 113……………………………………………………………

V． 病毒感染对细胞 miRNA 的调节 122………………………………………

VI． 细胞 miRNA 对病毒感染的调节 127………………………………………

VII． 结论 130………………………………………………………………………

参考文献 131………………………………………………………………………

使用 RNA 干扰对 HIV-1 感染的双重治疗

　 Julia J． M． Eekels 和 Ben BerkhoutI． RNAi: 从自然通路到治疗方法 142………………………………………

II． 抗病毒 RNAi 策略: HIV-1 的基因治疗 144………………………………

III． 定向到 HIV-1 RNA 基因组的什么位置? 147……………………………

IV． 组合 RNAi 方法 148…………………………………………………………

V． 定向到 HIV-1 复制的细胞辅因子 149……………………………………

VI． 临床前试验系统与安全性问题 152…………………………………………

VII． 临床试验引发的安全性问题 153……………………………………………

VIII． HIV-AIDS 的基因治疗试验 154……………………………………………

IX． 结论 155………………………………………………………………………

参考文献 155………………………………………………………………………

细胞网络失控: 在人病理学和治疗中 RNAi 机器的全局失调

　 Stefan Mockenhaupt、 Nina Schürmann 和 Dirk GrimmI． 简介: 谁来看守看守人? 166………………………………………………

II． 个体 miRNA 和 RNAi 组件的控制 170……………………………………

III． 在人类疾病中主要 RNAi 因子的全局失调 179……………………………

IV． 用宿主 RNAi 因子对病毒进行定量干扰 210………………………………

V． 基因 / RNAi 治疗研究的不良反应 218………………………………………

VI． 前景: RNAi 分子生物学与临床转化的相关性 229………………………

参考文献 231………………………………………………………………………

索引

彩图

Contents

Contributors..................................................................................... ixPreface............................................................................................. xi

Epigenetic Regulation of Gene Expression in HumanCells by Noncoding RNAs. . . . . . . . . . . . . . . . . . . . . . 1

Stuart Knowling and Kevin V. Morris

I. Introduction to ncRNA .................................................................. 1II. Epigenetics and ncRNAs................................................................ 3III. Long Intergenic Noncoding RNAs and Epigenetic Control of

Gene Transcription ....................................................................... 4References .................................................................................. 8

Small RNA-Induced Transcriptional Gene Regulation inMammals: Mechanisms, Therapeutic Applications,and Scope Within the Genome . . . . . . . . . . . . . . . . . . 11

Victoria A. Green and Marc S. Weinberg

I. Introduction ................................................................................ 12II. Mammalian Small RNA Biogenesis Pathways ..................................... 13III. Mechanisms of Small RNA-Induced Transcriptional Regulation ............ 16IV. Therapeutic Application of TGS ...................................................... 25V. The Scope of TGS Therapeutics ...................................................... 30

VI. Conclusions ................................................................................. 37References .................................................................................. 38

MicroRNA Function in the Nervous System . . . . . . . . . . 47

Roberto Fiore, Sharof Khudayberdiev, Reuben Saba, andGerhard Schratt

I. Introduction ................................................................................ 48II. miRNAs in Neural Differentiation ................................................... 49III. miRNAs in Postmitotic Neurons ...................................................... 60IV. The Roles of miRNA in Neurological Diseases ................................... 69

References .................................................................................. 91

v

The Role of MicroRNAs in Viral Infection . . . . . . . . . . . 101

Ashley P.E. Roberts, Andrew P. Lewis, andCatherine L. Jopling

I. Introduction ............................................................................... 102II. Regulation of miRNAs and Their Association with Disease.................. 108III. Introduction to Viral Infection ....................................................... 111IV. Viral miRNAs ............................................................................. 113V. Regulation of Cellular miRNAs by Viral Infection.............................. 122

VI. Regulation of Viruses by Cellular miRNAs ....................................... 127VII. Conclusions................................................................................ 130

References ................................................................................. 131

Toward a Durable Treatment of HIV-1 Infection UsingRNA Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Julia J.M. Eekels and Ben Berkhout

I. RNAi: From Natural Pathway to Therapeutic Method....................... 142II. Antiviral RNAi Strategies: Toward a Gene Therapy for HIV-1............. 144III. Where to Target the HIV-1 RNA Genome?..................................... 147IV. Combinatorial RNAi Approaches .................................................. 148V. Targeting Cellular Cofactors of HIV-1 Replication ............................ 149

VI. Preclinical Test Systems and Safety Concerns .................................. 152VII. Safety Issues Raised in Clinical Trials ............................................. 153VIII. Gene Therapy Trials for HIV-AIDS ............................................... 154IX. Conclusion................................................................................ 155

References................................................................................ 155

When Cellular Networks Run Out of Control:Global Dysregulation of the RNAi Machinery in HumanPathology and Therapy. . . . . . . . . . . . . . . . . . . . . . . . 165

Stefan Mockenhaupt, Nina Schurmann, and Dirk Grimm

I. Introduction: Quis Custodiet Ipsos Custodes or Who Watchesthe Watchmen?............................................................................ 166

II. Control of Individual miRNAs and RNAi Components........................ 170III. Global Dysregulation of Key RNAi Factors in Human Disease ............. 179IV. Quantitative Interference of Viruses with Host RNAi Factors ............... 210V. Adverse Effects in Gene/RNAi Therapy Studies................................. 218

vi contents

VI. Outlook: Relevance for Molecular Biology and Clinical Translationof RNAi ...................................................................................... 229References .................................................................................. 231

Index........................................................................................ 243

contents vii

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Ben Berkhout, Laboratory of Experimental Virology, Department of MedicalMicrobiology, Center for Infection and Immunity Amsterdam, AcademicMedical Center, University of Amsterdam, Amsterdam, The Netherlands(141)

Julia J.M. Eekels, Laboratory of Experimental Virology, Department of Med-ical Microbiology, Center for Infection and Immunity Amsterdam, AcademicMedical Center, University of Amsterdam, Amsterdam, The Netherlands(141)

Roberto Fiore, Interdisziplinares Zentrum fur Neurowissenschaften, SFB488Junior Group, Universitat Heidelberg, and Institut fur Neuroanatomie,Universitatsklinikum Heidelberg, Im Neuenheimer Feld 345, Heidelberg,Germany (47)

Victoria A. Green, Antiviral Gene Therapy Research Unit, Department ofMolecular Medicine and Haematology, University of the Witwatersrand,Johannesburg, South Africa (11)

Dirk Grimm, University of Heidelberg, Cluster of Excellence CellNetworks,Department of Infectious Diseases, Virology, Heidelberg, Germany (165)

Catherine L. Jopling, School of Pharmacy, Centre for Biomolecular Sciences,University of Nottingham, Nottingham NG7 2RD, United Kingdom (101)

Sharof Khudayberdiev, Interdisziplinares Zentrum fur Neurowissenschaf-ten, SFB488 Junior Group, Universitat Heidelberg, and Institut furNeuroanatomie, Universitatsklinikum Heidelberg, Im Neuenheimer Feld345, Heidelberg, Germany (47)

Stuart Knowling, Department of Molecular and Experimental Medicine,The Scripps Research Institute, La Jolla, California, USA (1)

Andrew P. Lewis, School of Pharmacy, Centre for Biomolecular Sciences,University of Nottingham, Nottingham NG7 2RD, United Kingdom (101)

Stefan Mockenhaupt, University of Heidelberg, Cluster of ExcellenceCellNetworks, Department of Infectious Diseases, Virology, Heidelberg,Germany (165)

Kevin V. Morris, Department of Molecular and Experimental Medicine,The Scripps Research Institute, La Jolla, California, USA (1)

Ashley P.E. Roberts, School of Pharmacy, Centre for Biomolecular Sciences,University of Nottingham, Nottingham NG7 2RD, United Kingdom (101)

ix

Reuben Saba, Interdisziplinares Zentrum fur Neurowissenschaften, SFB488Junior Group, Universitat Heidelberg, and Institut fur Neuroanatomie,Universitatsklinikum Heidelberg, Im Neuenheimer Feld 345, Heidelberg,Germany (47)

Nina Schurmann, University of Heidelberg, Cluster of Excellence CellNet-works, Department of Infectious Diseases, Virology, Heidelberg, Germany(165)

Gerhard Schratt, Interdisziplinares Zentrum fur Neurowissenschaften,SFB488 Junior Group, Universitat Heidelberg, and Institut fur Neuroana-tomie, Universitatsklinikum Heidelberg, Im Neuenheimer Feld 345,Heidelberg, Germany (47)

Marc S. Weinberg, Antiviral Gene Therapy Research Unit, Department ofMolecular Medicine and Haematology, University of the Witwatersrand,Johannesburg, South Africa (11)

x contributors

Preface

Despite centuries filled with biological discoveries that shaped our funda-mental view and understanding of nature and that refined our approaches todistinguish and treat human diseases, few findings made such a pivotal dualimpact as the one that RNA interference (RNAi)—an evolutionarily conservedcellular means of specific and potent gene silencing—is active in mammals.Indeed, what we have witnessed over the past decade since RNAi’s initialdescription in a nematode is nothing short of a revolution in our awareness ofthe amazing intricacy underlying gene regulation in higher organisms, and yetwe are probably only beginning to appreciate the inherent beauty and complexdiversity of the numerous relevant mechanisms. Concurrently, we are experi-encing constant shifts in our perception of the molecular processes governinghuman diseases that offer a myriad of exciting and promising novel avenues toexpand our repertoire of diagnostic and prognostic tools and to further enlargeour clinical arsenal of potent intervention strategies.

In this issue of Progress in Molecular Biology and Translational Sciences, acollection of six chapters provides an insight into the amazing sophisticationand versatility underlying ‘‘cellular RNAi mechanisms’’ in healthy or diseasedhumans, hoping to leave the reader with an impression of the sometimesbreathtaking speed at which the entire field is moving and incessantly pushingthe boundaries of biology and medicine like no other before.

Starting off the issue is a chapter by Stuart Knowling and Kevin V. Morris,which reviews one of the latest and most unanticipated discoveries in our area,namely the fact that small RNAs not only regulate gene expression on theposttranscriptional level (the canonical RNAi pathway) but also exert theircontrol at the stage of transcription. Even more, we are just now starting torealize that small RNAs not only suppress but can also activate gene expression,by means that still escape our current understanding but will certainly provideintriguing research topics for years to come. As is typical for this review seriesas well as for the field of RNAi, Knowling and Morris finally round up theirchapter by highlighting the promises to translate these new discoveries intonovel future biomedical concepts ideally allowing us to control human geneexpression at yet another important level.

Following up on this introductory overview is a second complementingchapter by Victoria Green and Marc Weinberg, which delves even deeper intothe astonishingly large variety of molecular mechanisms that potentially govern

xi

these noncanonical epigenetic RNAi-related regulatory processes in humancells. While today we can hardly fully appreciate the surprisingly vast array ofnoncoding RNA-based networks acting at this fundamental level of geneexpression, Green and Weinberg already present their insightful vision of thesignificant possible implications of these new findings for our basic understand-ing of the function of human cells as well as for the implementation of entirelynovel biomedical strategies in future drug and gene therapy development.

Leaving the cellular nucleus and entering gene regulation in the cytoplasm,Roberto Fiore, Sharof Khudayberdiev, Reuben Saba, and Gerhard Schratt nextreview another flourishing and exciting area of current RNAi research which isposttranscriptional gene silencing by miRNAs in the central nervous system.Also here, our understanding of how RNAi shapes and controls central cellularprocesses such as neuronal differentiation, synaptogenesis and plasticity is in itsinfancy at best, as are our insights into how RNAi is involved in higher cognitivefunctions and the etiology of neurological diseases. All these are utmostfascinating and captivating questions that the authors will address and whoseanswers will continue in the following years to revolutionize and overthrow ourcurrent picture of cellular RNAi mechanisms in the human nervous system inhealth and disease.

Complementing these three reviews of endogenous RNAi processes inhuman cells, two consecutive chapters then enter the thrilling field of patho-genic viruses and assess their relationship with cellular RNAi mechanisms fromthe molecular biology and translational sciences standpoints. In the first, AshleyP.E. Roberts, Andrew P. Lewis, and Catherine L. Jopling will initially provide abroad and comprehensive overview over virus structure and function, in gen-eral, before moving into the incredible intricacies of virus–host interactionsinvolving miRNAs and human RNAi machinery. One special example that willthen be discussed in particular detail is the interplay of human hepatitis C virus(HCV) with a liver-specific miRNA the unconventionality of which has consis-tently amazed the RNAi and virus communities over the past few years and willhopefully excite the readers of this chapter as well.

Taking over from this overview over how viruses exploit and modulate thehuman RNAi machinery, Julia Eekels and Ben Berkhout next highlight howone can turn this cellular mechanism against the intruder and develop clinicalRNAi-based strategies that not only aim to battle and eradicate the AIDS virusHIV-1 but concurrently also rob it of any chance to ever escape from therapy bynatural mutation. Therefore, the authors first thoroughly review the plethora ofstrategies and tools that have recently been discussed, developed, and validatedto achieve these goals, before they describe combinatorial RNAi targeting ofmultiple essential viral and cellular genes as the latest and most promisingtherapeutic approach to tackle and forever eliminate the HIV pathogen frominfected patients.

xii preface

Finishing off this issue is a chapter from our own group that once againdocuments the remarkably tight affiliation of basic and applied RNAi researchby reviewing our emerging picture of cellular RNAi mechanisms potentiallydysregulated in human cancers and other devastating diseases, and by concom-itantly discussing the novel therapeutic concepts and translational avenues thatfortuitously continue to surface and materialize with each new report. Extend-ing the preceding chapters, special focus will also be put on viruses not onlybecause they represent beautiful authentic examples for molecular means toperturb, hijack, and reprogram cellular processes but because viruses are alsobeing engineered as RNAi-expressing tools to treat human diseases that canoccasionally turn into our worst enemy and unintentionally overthrow thehuman RNAi machinery. The related recent observations and vital lessonslearned will be discussed, before Stefan Mockenhaupt, Nina Schurmann, andDirk Grimm round up the chapter and issue with an outlook into what may bethe next important steps to even further advance our understanding andutilization of cellular RNAi mechanisms and to ensure that the whole fieldwill positively continue to prosper and thrive for many more years to come.

Dirk Grimm

preface xiii

Epigenetic Regulation of GeneExpression in Human Cells byNoncoding RNAs

Stuart Knowling and KevinV. Morris

Department of Molecular and ExperimentalMedicine, The Scripps Research Institute,La Jolla, California, USA

I. Introduction to ncRNA ...... ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... 1II. Epigenetics and ncRNAs ....... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... 3III. Long Intergenic Noncoding RNAs and Epigenetic Control of Gene

Transcription .... ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... 4References..... .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... 8

Emerging evidence has begun to suggest that a vast array of noncodingRNAs is operative in human cells, with some containing the ability to directlymodulate gene transcription. While observations of noncoding-RNA-basedepigenetic regulation of gene expression were in the past relegated toimprinted or X-linked genes, it is now becoming apparent that several differentgenes in differentiated cells may be under some form of RNA-based regulatorycontrol. Studies have begun to discern certain aspects of an underlying mecha-nism of action whereby noncoding RNAs modulate gene transcription. Muchof the evidence suggests that noncoding RNAs are functional in controllinggene transcription by the targeted recruitment of epigenetic silencing com-plexes to homology-containing loci in the genome. The results of these studies,as well as the implications that a vast array of noncoding-RNA-based regulatorynetworks may be operative in human cells, are discussed. Knowledge of thisemerging RNA-based epigenetic regulatory network has implications in cellu-lar evolution as well as in an entirely new area of pharmacopeia.

I. Introduction to ncRNA

The central dogma of molecular biology states that DNA sequence infor-mation tends to flow in one direction from a protein-coding region of thegenome, through transcription to mRNA, and then, finally, is translated toprotein. Every step along the way is assisted by proteins; therefore it is easyto see why it is described as a protein world. Recently though, new techniquessuch as genomic tiling arrays and cDNA sequencing, as used in RNA Seq, have

Progress in Molecular Biology Copyright 2011, Elsevier Inc.and Translational Science, Vol. 102 1 All rights reserved.DOI: 10.1016/B978-0-12-415795-8.00003-9 1877-1173/11 $35.00

provided a surprising view of how little of the human genome is actually usedfor protein coding (� 2% of the eukaryotic genome encodes protein-codinggenes—mRNA). The advent of these new DNA sequencing technologies hasallowed RNA transcription in eukaryotic genomes to be assessed with higheraccuracy and better resolution than ever before. The ENCODE (encyclopediaof DNA elements) project used deep sequencing of human cells to identify allfunctional elements in the human genome sequence by the analysis of a smallfraction of RNAs (< 200 nucleotides—nt).1 Approximately 90% of the genomeis transcribed,2 generating a large number of RNA transcripts that are non-protein-coding RNAs (ncRNAs). The results of ENCODE show that RNA canbe processed via an unknown mechanism to yield complex populations of bothshort and long RNAs, which overlap both the 50 and 30 ends of protein-codingtranscripts.1

The procession of longer RNA to shorter RNA to yield unique secondaryand tertiary structures that may participate in cellular processes has long beenknown in the presence of3 and absence of protein.4,5 This knowledge iscurrently being exemplified in cellular and pharmaceutical processes by RNAaptamers.6 The production of these long or short ncRNAs effectively splitsDNA transcription into two camps: coding and noncoding transcripts,7 wheretraditional noncoding transcripts are viewed to be ribosomal RNA (rRNA) andtransfer RNA (tRNA), both of which play essential functions in protein trans-lation. Although there is disagreement as to whether these ncRNAs are tran-scriptional ‘‘noise,’’ there is growing evidence that ncRNAs (miRNAs, smallRNAs) play an important role in cellular function7 as is the case for theaforementioned tRNA and rRNA. Therefore, ncRNA raises the enticing possi-bility that the large number of ncRNAs being discovered play key roles incellular control. Recently, evidence has begun to emerge suggesting thatncRNAs play a role in controlling gene transcription through the targetedrecruitment of epigenetic silencing complexes to particular loci (reviewed inRefs. 8,9) and through degradation of transcriptionally active mRNAs viaSTAU-1-mediated RNA decay.10 Staufen 1 is a protein that binds to double-stranded RNA and mediates (STAU1)-mediated messenger RNA decay(SMD). SMD degrades active mRNAs that contain 30 untranslated regions(30UTRs), which contain a STAU1 binding site (of approximately 688 nucleo-tides and consisting of a 19-base pair stem with a 100-nt apex).11 Although theSTAU1 binding site is not present in all the 30UTRs of all mRNAs, it wasrecently shown that a cytoplasmic, polyadenylated lncRNA (ncRNA 1/2-sbsRNAs) that contains Alu elements can form imperfect base pairing withAlu elements in the 30UTR of mRNA, forming an STAU-1 binding site and thustargeting it for SMD. Interestingly, the ncRNA 1/2-sbsRNAs was found inevery tissue examined by Gong et al.10 but only in the cytoplasmic fractionsof HeLa cells. 1/2-sbsRNA1 was found not to be a substrate for the enzymes di.

2 KNOWLING AND MORRIS

cer 1 (DICER1) or argonaute 2 (AGO2), and thus represents an unexpectedstrategy that cells can use to recruit proteins to mRNAs and facilitate theirdecay. Only 23% of ncRNA contain Alu elements, so it is possible that furtherpathways exist for regulation of dsRNA by dsRNA-binding proteins.

II. Epigenetics and ncRNAs

Epigenetics is the study of the underlying changes in phenotype that arecaused by alterations to the expression of the genome by chemical modificationof the DNA molecule but without changing the overall DNA sequence.Although several forms of epigenetic regulation exist, the two main forms ofinterest for this review are the (1) addition of chemical groups to specific bases,as with DNA methylation and (2) the local alterations of histones, such astargeted methylation at particular lysines that affects the accessibility of thesurrounding genomic DNA to the transcriptional machinery. Other modifica-tions of interest to this review are the splicing of RNA to form novel structuresand double-stranded RNA, as with RNA interference (RNAi) pathways.12

The current view of genetics is dominated by Darwinism, but epigeneticcontrol of the genome offers a ‘‘touch of soft Lamerckism’’7 in that epigeneticchanges caused by environmental processes are capable of being passed on todaughter cells. It has been shown that epigenetic changes to the IGF2 gene thatwere caused by prenatal exposure to the Dutch Hunger Winter in the1944–1945 famine are persistent across familial generations six decadeslater,13 an effect that has also been noted in mice.14,15 The prospect of theability to impact further generations of genome via epigenetic methods runcounter to Darwinian existence but suggests that environmental changesindeed affect the expression of DNA and can be passed onto offspring. Thecomprehensive role that ncRNA plays in this epigenetic control remains to beseen, but if the recent observations are any indication from a limited number ofstudies,16–23 the notion is that much of the ncRNAs in human cells might beactive regulators involved in controlling gene expression via the targetedrecruitment of epigenetic complexes to various loci in the genome.

The obvious question is whether there is a link between the large amount oftranscribed ncRNA and the regulation of genome modification via epigenetics.The fact that almost four times the amount of ncRNA is transcribed, comparedto protein-coding RNA,24 suggests there must be a function for these ncRNAs:that is, this is an immense cost to the cell with regard to energy expenditure.Expressed ncRNAs show clear evolutionary conservation25 and many emanatefrom gene promoter regions, which tend to be more conserved than protein-coding genes,26 thus suggesting a level of retention in the machinery of the celland a possible role in gene level control, possibly via the recruitment of

EPIGENETIC REGULATION OF GENE EXPRESSION IN HUMAN CELLS 3

transcriptional factors to these regions. It should be noted that, althoughncRNAs can be found in both polyadenylated and unadenylated (� 50% oftranscriptome)27 forms and do not contain classical ORFs longer than 100amino acids, some may in fact encode small peptides.28 Although we currentlycannot exclude the possibility that the mere act of transcription of noncodingDNA is more important than the resultant ncRNA, there is steadily growingevidence that at least some of these molecules play specific roles in eukaryoticcells and gene expression (reviewed in Ref. 8).

III. Long Intergenic Noncoding RNAs and Epigenetic Control ofGene Transcription

Long intergenic noncoding RNAs (lincRNAs) are a heterogeneous group oftranscripts involved in epigenetic control of the cell, ranging in size from � 300nucleotides to the thousands. Currently, the human catalog of lincRNAs isthought to be around 3300, although the true number may be closer to 4500.29

The most studied lincRNAs to date include Xist RNA, a 19,000-nucleotideuntranslated transcript that coats the X-chromosome from which it istranscribed and acts in cis to cause inactivation due to the loss of histonemodification by acetylation and methylation.30 AIR is another long ncRNAthat acts in cis to interact with the promoter chromatin and the H3K9 histonemethyltransferase G9a to cause gene suppression via hypermethylation.31

There is also the well-characterized, 2200-nucleotide lincRNA, HOTAIR, atrans-acting, long, intergenic ncRNA located on the HOXC locus that interactswith the polycomb repressive complex 2 (PRC2, a histone 3 methyltransferaseinvolved in gene silencing) and subsequent trimethlyation of HOXD.32

HOTAIR expression levels increase approximately 125-fold during the progres-sion of breast cancer, eventually leading to altered gene expression, specificallyepigenetic silencing of tumor suppressor genes.33

As well as imprinting roles, lincRNA can also play an important role in thestructure of the cell as exemplified by NEAT1 RNA,34 a 4-kb lincRNA that isretained in the nuclei and localizes to paraspeckles which are present through-out interphase, indicating a role in nuclear function.35 Others have found thatlincRNAs are involved in structural characteristics of chromatin.36

Taken together, all these lincRNAs are vital for cellular survival and differ-entiation as well as organismal development. These are the traditional imprint-ing genes that have been well studied and have provided the first concreteevidences that lincRNAs can play a role in gene expression (reviewed in Ref.37). One thing in common with these loci is that they all utilize the endogenousepigenetic regulatory machinery in their regulation of the particular targeted

4 KNOWLING AND MORRIS

loci. Unfortunately, these complex imprinted loci, as well as the role lincRNAsplay in their respective regulation, have been somewhat overlooked by thegeneral scientific community. The implications of this body of work have beenrelegated to a peculiar aspect of cellular differentiation. A recent body ofevidence has emerged, suggesting that lincRNAs can play a role in the regula-tion of gene transcription in differentiated cells.21 These observations of anendogenous mechanism involved in the control of gene transcription offersignificant insights into the possibility of specifically controlling protein-codinggenes at the transcriptional level. Such an opportunity may provide the frame-work for a new class of pharmacopeia.

Recent work involving mouse cells has shown that the Oct-4 and Nanogtranscription factors directly target and regulate the transcriptional expressionof two lncRNAs.38 Further study in human cells has shown that Oct-4 is furtherregulated by the action of a psuedogene-expressed antisense ncRNA thatregulates the transcription of Oct-4.19 Interestingly, these ncRNAs were notonly antisense and overlapping the Oct-4 promoter but also un-polyadenylatedand found to regulate Oct-4 transcription by the targeted recruitment ofvarious epigenetic remodeling proteins.19 The results for the ncRNA regulationof Oct-4 reflect those observed for the respective antisense ncRNAs associatedwith p15, p21, and HOX loci.21,23,32 Based on these bodies of work, a model hasbeen developed, wherein the long antisense ncRNAs are expressed either in cisor trans and function in trans to both target homology-containing loci as well asrecruit particular proteins to the homology-containing targeted loci (Fig. 1).Such a model implies that the role for long ncRNAs is to both bind epigeneticremodeling proteins as well as to guide them to appropriate loci in the genomeand instigate epigenetic remodeling (Fig. 1).

Not all lincRNAs appear to regulate their sense counterpart in a discordantmanner. It has been proposed on the basis of the RIKEN consortium analysisof RNA-based regulation in mouse cells (Functional Annotation Of Mammali-an Genome, FANTOM) that another form of ncRNA-based regulation isongoing, termed concordant regulation.39 Concordant regulation was observedwhen antisense ncRNAs were degraded by RNAi, and the net result was theactivation of the ncRNA-targeted gene. It has been proposed by Wahlestedtthat concordant regulation was functional via an RNA/RNA interaction in thecytoplasm and, thus, when the RNA/RNA interactions or stoichiometric ratiosare altered by the action of RNAi, the result is increased sense/mRNA expres-sion and gene activation (personal communication based on Ref. 39). Recentstudies carried out with the gene Nanog, also involved in stem cell genesis,determined that, like Oct-4,19 there are also Nanog-specific psuedogenes,which express long antisense ncRNAs. Interestingly, similar to the Oct-4ncRNAs, the Nanog ncRNAs also lacked a polyadenylated tail but, unlikeOct-4 ncRNAs, they did not appear to share homology with the Nanog

EPIGENETIC REGULATION OF GENE EXPRESSION IN HUMAN CELLS 5

promoter. When these antisense ncRNAs were suppressed, concordant regula-tion of Nanog was observed.40 Discordantly regulated genes are regulated in theinverse manner to concordantly regulated genes and are reviewed in Refs. 19, 41.

Several insights have been obtained from these works. Taken together,the Oct-4 discordant and Nanog concordant model systems might be tellingus something important about endogenous RNA circuits and gene regulation.No doubt, when one considers now the plethora of ongoing RNA interactionsin the cell, such as these observations whereby psuedogene-expressed anti-sense ncRNAs are actively regulating their respective mRNA-expressingcounterpart in either a positive or negative manner, it indicates a far morecomplex landscape of RNA-based regulation than has been previouslyappreciated.

It is now clear that there is significant and growing evidence that small andlong antisense ncRNAs are able to direct the epigenetic control of genetranscription. Such a model was recently proposed for the PRC2-associatedlincRNA TUG1,29 a gene that is upregulated in response to DNA damage

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H3K27AcetylH3K27AcetylH3K27me3

H3K27 H3K9AcetylH3K9Acetyl

H3K9me2

H3K9

Suv39H1 ?

G9a ?

HDAC-1

RNAP IIAgo1

DNMT3A

EZH2?

H3K9me2

A

C

D

E

F

B

CpG

CpG

CpGCpG

H3K

9me2

H3K27me3H

3K27m

e3

3�

5�

FIG. 1. Endogenous ncRNA-directed epigenetic regulation of gene transcription. (A) Longantisense ncRNAs can be expressed in cis or (B) trans, where they (C) fold into complex structuresthat can (D) contain both target homology to other loci or the bidirectionally transcribed loci in thegenome as well as possess an ability to bind epigenetic remodeling proteins. The ncRNA-directedepigenetic complexes could then (E) target the silencing of the corresponding homology-containingloci, which can result in (F) heterochromatinization of the targeted site. (See Color Insert.)

6 KNOWLING AND MORRIS

through the tumor suppressor gene p53. Knockdown of TUG1 by siRNA led toan upregulation of 120 genes, suggesting that PRC2 and TUG1 (and otherlincRNA) repress transcription via directing silencing to specific loci in the cell.

It has also been noted that, although many lincRNAs act in a repressivemanner by chromatin modification, it is possible that many genes couldbe upregulated in a similar manner by chromatin modification42; this seems aplausible occurrence based on the striking number of observations to datedemonstrating RNA-based regulation of gene expression (reviewed in Ref.43). Such a notion runs counter to the dogma arguing that transcription ispredominantly controlled by transcription factors and other DNA-bindingproteins. However, it may also be that the localization of transcription factorsand larger protein-activating complexes at particular loci in the genome alsorequire the action of lincRNAs. This has been suggested recently for thep53-activated lincRNA, lincRNA-p21.44 The promoter region of lincRNA-p21 contains a high number of p53-binding motifs and is significantly inducedupon p53 activation. Upon activation, lincRNA-p21 plays a significant role inapoptosis of the cell, acting as a downstream repressor in p53 transcriptionalresponse. lincRNA-p21 was shown to interact with the DNA-binding proteinhn-RNP-K, indicating that lncRNA may alter the binding specificity ofDNA-binding proteins in order to alter their target specificity, subsequentlyaltering gene expression.

Regardless, recent observations have begun to craft a modified version ofpreviously held dogma. This new model intercalates the action of ncRNAs inthe control of gene expression and is based on a growing body of evidence.Such observations suggest only one plausible notion: that there are multiplelayers of RNA-based regulation, be they direct RNA/RNA-, RNA/prote-, orRNA/DNA-based. The basal strata of RNA-directed regulation of gene expres-sion are most likely at the act of transcription, as the RNA requires transcrip-tion in its essence of function. This basal layer of RNA-based regulation mayhave some striking similarities to and functional properties involved in themechanism that dictates euchromatin versus heterochromatin transitions inthe nucleus. Or, alternatively, they have the same mechanism of action. Cer-tainly, work with imprinted genes and X-inactivation, which involve significantcompaction of the ncRNA-targeted loci, would suggest that many similarcomponents are involved in the basal modes of action.37,45

The regulation of transcription, via ncRNA-targeted epigenetic controllingmechanisms, might in fact be the essence of endogenous gene regulation andthe fundamental core regulator of protein production. One could envision thatthe stoichiometric ratios of particular ncRNAs are active in regulating genetranscription, but also that these ncRNAs are themselves regulated byother ncRNAs and RNA-binding complexes. Such a concept adds so manylayers of complexity that one has to envision the cell as a larger ‘‘ecosystem’’ of

EPIGENETIC REGULATION OF GENE EXPRESSION IN HUMAN CELLS 7

interactions. While such speculation cannot in principle be experimentallyvalidated, as the complexity within the cell would no doubt be infinite, thereare some aspects of this regulation that are noteworthy: namely, the notion thatmodulating the expression paradigm of particular ncRNA regulatory networksmight allow an ability to modulate both single gene/protein expression andpossibly yet-to-be-discovered larger complex networks regulated by a single ora few ncRNAs or the particular single gene/protein. Such knowledge could intheory allow increased gene-specific targeting capabilities to be developed aswell as studies on larger gene regulatory networks to be carried out. No doubt,we will, in the not too distant future, be faced with the notion that largersystems have the capability of functioning in an autonomous and uniquemanner far different than each of the constituent parts. Such a notion willprovide information on the essence of gene regulation and possibly some cluesas to how natural selective pressures are operative in living systems on genomicevolution.

Acknowledgments

The Morris lab is funded by Grants NIH R01 HL083473, NIH R01 AI084406, NIH R01CA151574, and NIH R01 CA153124.

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Small RNA-InducedTranscriptional GeneRegulation in Mammals:Mechanisms, TherapeuticApplications, and ScopeWithin the Genome

Victoria A. Green andMarc S. Weinberg

Antiviral Gene Therapy Research Unit,Department of Molecular Medicine andHaematology, University of theWitwatersrand, Johannesburg, South Africa

I. Introduction .... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 12II. Mammalian Small RNA Biogenesis Pathways.... ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 13

A. Canonical miRNA Biogenesis ..... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 13B. Noncanonical miRNA and siRNA Biogenesis .... .. ... .. ... .. ... .. ... .. .. ... .. ... 15

III. Mechanisms of Small RNA-Induced Transcriptional Regulation ..... .. .. ... .. ... 16A. Epigenetic Changes Associated with TGS....... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 16B. Promoter-Associated Transcripts ..... ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 18C. Transcriptional Interference.... ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 19D. Transcriptional Gene Activation .... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 23

IV. Therapeutic Application of TGS ...... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 25A. Duration and Potency of TGS ...... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 25B. Expressed TGS Effectors ..... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 26C. Disease Treatment .... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 27D. Targeting Nonpromoter Regions ..... ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 29

V. The Scope of TGS Therapeutics ...... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 30A. Tissue Specificity ...... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 30B. Promoter Target Design.... ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 30C. Promoter Architecture and Gene Susceptibility ...... .. ... .. ... .. ... .. .. ... .. ... 33D. Inducing a TGS-Permissive State .... ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 36

VI. Conclusions ..... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 37References ...... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... 38

Argonaute-bound small RNAs, derived from RNA interference and relatedpathways, are well-known effectors of posttranscriptional gene silencing(PTGS). Yet, these complexes also play an important role in affecting geneexpression at the transcriptional level, either by transcriptional gene silencing(TGS) or activation (TGA). Our current understanding of how small RNAs areable to both activate and suppress transcription is unclear. In this review, webriefly outline the biogenesis of small RNAs and explore the mechanismsbehind the various phenomena attributed to AGO-bound small RNA-mediated

Progress in Molecular Biology Copyright 2011, Elsevier Inc.and Translational Science, Vol. 102 11 All rights reserved.DOI: 10.1016/B978-0-12-415795-8.00005-2 1877-1173/11 $35.00