药悟
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Mouldy Sioud
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The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.
通过将外源双链 RNA 引入细胞而使基因表达沉默的发现，揭示了一个通过各种小 RNA 途径调控基因表达的隐藏层面。这些小 RNA 途径不仅参与调控内源性基因的表达，还能防御病毒感染，并在转录后和表观遗传水平上保护基因组免受侵入转座子的影响。所有内源性 RNA 干扰途径都共享一种保守的效应复合物，该复合物至少含有一种精氨酸蛋白和一条短的单链 RNA。这种精氨酸蛋白-RNA 复合物可以抑制基因的转录、切割特定位置的 mRNA 或阻止 mRNA 翻译成蛋白质。本综述概括了 RNAi 发现的历史背景、功能作用和作用机制，并对比介绍了 CRISPR 干扰技术。
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RNA silencing is a highly conserved gene regulation mechanism in which small RNA molecules silence gene expression by either suppressing transcription (transcriptional gene silencing) or by triggering a sequence-specific degradation of target mRNA, a process known as posttranscriptional gene silencing or RNA interference (RNAi). Although Fire’s Nature paper is considered a key breakthrough in the history of RNAi technology, it should be noted that in 1990 researchers described for the first time that RNA could potentially suppress gene expression in plants. Indeed, Napoli and Jorgensen were the first to report an RNAi type of phenomenon in plants 1990. By overexpressing the gene for chalcone synthase, an enzyme responsible for the production of anthocyanin pigments, the authors wanted to generate more purple transgenic petunia flowers. Surprisingly, some of the transgenic petunia lost both endogenous and transgene expression. This phenomenon was described as “cosuppression” and can be induced by single copy transgene that integrates into the genome in multicopy arrays. Later in 1992, a similar phenomenon was observed by Romano and Macino in Neurospora crassa.
RNA 沉默是一种高度保守的基因调控机制，小 RNA 分子可以通过抑制转录（转录基因沉默）或引发靶标 mRNA 特异序列降解（称为转录后基因沉默或 RNA 干扰）的方式来沉默基因表达。虽然 Fire 在 Nature 杂志上发表的论文被视为 RNA 干扰技术的关键突破，但我们需要注意到，早在 1990 年研究人员就首次报道了 RNA 可能会抑制植物中的基因表达。事实上，Napoli 和 Jorgensen 在 1990 年首次报告了植物中的 RNA 干扰现象。他们通过过度表达负责花青素合成的黄酮合成酶基因，试图培育出更多紫色的转基因矮牵牛花。出乎意料的是,一些转基因矮牵牛不但失去了外源基因的表达,内源基因的表达也被抑制了,这种现象被称为"共抑制"。这种现象可能是由整合到基因组中形成多拷贝阵列的单拷贝转基因引起的。后来在 1992 年,Romano 和 Macino 在神经孢子菌中也观察到了类似的现象。
In 1995, Guo and Kemphues were the first to report RNA silencing in animals. The authors reported that sense RNA was as effective as antisense RNA for suppressing the expression of par-1 gene in worm. Par-1, a gene required for establishing polarity in Caenorhabditis (C) elegans . At that time, antisense was one of the most attractive strategy for inhibiting gene expression. As a control, the authors used the sense par-1 RNA that would not hybridize to par-1 transcript. Interestingly, the degradation of par-1 mRNA was still observed. Although the results were interesting, the mechanism of mRNA degradation remained unknown.
在 1995 年，Guo 和 Kemphues 首次报告了动物中的 RNA 沉默现象。作者报道说，对于抑制线虫中par-1基因表达，正义 RNA 和反义 RNA 一样有效。Par-1是秀丽隐杆线虫（Caenorhabditis elegans）中建立极性所需的基因。当时，利用反义 RNA 抑制基因表达是最受关注的策略之一。作为对照，作者使用了不与 par-1 转录本杂交的正义 par-1 RNA。有趣的是，依然观察到了 par-1 mRNA 的降解。尽管结果很有意义，但 mRNA 降解的机制仍然是未知的。
In 1998, Fire and Mello showed in the worm C. elegans that double-stranded RNA (dsRNA), but not single-stranded RNA (ssRNA), was involved in gene silencing and coined the term “RNA interference, RNAi”. The authors thought that the results of Guo and Kemphues showing that introduction of sense RNA leads to gene silencing could have been due to the contamination of the RNA preparation by dsRNA resulting from the activity of bacteriophage RNA polymerases. They purified the sense and antisense RNAs, then directly compared their effects on the unc-22 gene, one of a set genes identified using classical genetics that affect muscle function in C. elegans. Overall, their data showed that exogenously introduced dsRNA reduces the expression of homologous mRNAs in C. elegans, and that RNAi may have a catalytic component. While these findings establish a new concept for the effects of RNA on gene silencing by highlighting a role for dsRNA, the mechanism of unwinding dsRNAs to promote the degradation of target mRNAs remained unresolved. It was later discovered that dsRNA molecules were processed into shorter intermediates dsRNAs and then into single-stranded RNAs that can ultimately base pair to the mRNA targets and induce their cleavage. These RNA intermediates were called small interfering (si) RNAs and proved to be the main effectors of RNAi induction in mammalian cells. In conclusion, in RNAi, a dsRNA is involved and may originate from viral infection (exogenously), inverted-repeat transgenes, or from transcription of nuclear genes (endogenously).
1998 年，Fire 和 Mello 在秀丽隐杆线虫中证实，是双链 RNA（dsRNA）而非单链 RNA（ssRNA）参与基因沉默，并创造了“RNA 干扰（RNAi）”一词。作者认为，之前 Guo 和 Kemphues 观察到引入同义 RNA 导致基因沉默的现象，可能是由于 RNA 制备过程中存在 dsRNA 污染所导致的，这种污染是由细菌病毒 RNA 聚合酶活性造成的。为了验证这一假设，他们纯化了正义和反义 RNA，直接比较了它们对 unc-22 基因的影响，该基因是经典遗传学鉴定的影响秀丽隐杆线虫肌肉功能的基因之一。总的来说，他们的数据表明，外源引入的 dsRNA 可以降低秀丽隐杆线虫中同源 mRNA 的表达，并且 RNAi 可能具有催化性质。尽管这些发现通过突出 dsRNA 的作用建立了 RNA 对基因沉默的新概念，但解开 dsRNA 从而诱导靶标 mRNA 降解的具体机制当时还未阐明。随后的研究发现，dsRNA 分子被加工成较短的中间 dsRNA，然后进一步加工成能够与 mRNA 靶点形成碱基配对并诱导其降解的单链 RNA。这些 RNA 中间体被称为小干扰(si)RNA，在哺乳动物细胞中证明是 RNAi 诱导的主要效应因子。综上所述，RNAi 机制中牵涉到 dsRNA 的参与，这些 dsRNA 可能来源于病毒感染(外源性)、反向重复转基因或来自内源性核基因的转录。
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At the beginning, the use of RNAi was limited to flies, worms, and plants, as the introduction of long dsRNA into mammalian cells induces an interferon response that triggers a general inhibition of translation, abrogating the specificity of RNAi. However, in 2001 Tuschl and colleagues showed that duplexes of 21-nucleotide RNAs can bypass the interferon pathway and induce the degradation of their specific mRNA targets and thereby inhibiting the resulting protein expression. These experiments were performed in cancer cell lines that may lack the receptors that recognize small RNAs. In this respect, several groups were able to show that certain siRNA sequences are potent activators of the immune system . The activation of innate immunity by chemically made siRNAs is sequence-dependent and is normally mediated by Toll-like receptors (TLRs). We and others have demonstrated that the ribose sugar backbone are both necessary and sufficient for TLR7 and TLR8 recognition and that short ssRNA only requires several uridines in close proximity to render them effective immunostimulators. Fortunately, modifying siRNAs with as few as two 2′-OMe modifications at any position in the siRNA can eliminate the immunogenicity that can occur with unmodified siRNAs in immune cells. Other 2′-ribose modifications such as 2′-F and 2′-deoxy can also reduce immune activation; however, the effects are not as pronounced as 2′-OMe RNAs. Similarly, unmodified CRISPR guide RNAs induced type I interferon production in vitro in peripheral blood mononuclear cells , and that substitution of 2′OMe residues eliminated this response. Interestingly, 2′-modified RNAs can function as TLR antagonists.
早期，RNAi 技术仅限于果蝇、线虫和植物，因为向哺乳动物细胞引入长双链 RNA 会诱导干扰素反应，该反应会触发翻译的普遍抑制，从而消除 RNAi 的特异性。然而，2001 年，Tuschl 及其同事证明 21 核苷酸的双链 RNA (siRNA) 可以绕过干扰素通路，并诱导其特异性 mRNA 靶标降解，从而抑制相应蛋白质的表达。这些实验是在癌细胞系中进行的，癌细胞系可能缺乏识别小 RNA 的受体。在这方面，一些研究团队能够证明某些 siRNA 序列是免疫系统的强效激活剂。化学合成的 siRNA 激活先天免疫具有序列依赖性，通常由 Toll 样受体 (TLRs) 介导。我们和其他研究人员已经证明，核糖的糖链骨架对于 TLR7 和 TLR8 的识别既是必需的也足够，并且短单链 RNA 只需要几个紧密排列的尿苷就能使其成为有效的免疫刺激因子。幸运的是，用少量 (只需两个) 2'-O 甲基 (2'-OMe) 修饰 modification 修饰 siRNA 就可以消除未修饰 siRNA 在免疫细胞中可能产生的免疫原性。其他 2'-核糖修饰物 (例如 2'-F 和 2'-脱氧) 也能降低免疫激活，但效果不如 2'-O 甲基 RNA 显着。类似地，未修饰的 CRISPR 引导 RNA 在体外外周血单核细胞中诱导 I 型干扰素产生，而替换为 2'-O 甲基残基则消除了这种反应。有趣的是，2'-修饰的 RNA 还可作为 TLR 拮抗剂发挥作用。
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Early in vitro work by Hannon, Zamore, Tuschl, and colleagues showed that the RNAi active protein complex contains 25 nt-long small RNAs that were homologous to the target mRNAs. Shortly thereafter, several laboratories were able to identify enzymes and cofactors in RNAi pathways using genetic and biochemical methods. These efforts revealed that dsRNAs are cleaved by the enzyme Dicer, a RNase III family member, to generate 21–23-nt shorter RNA duplexes (Fig. 1). Dicer is a conserved protein among eukaryotes and can have multiple homologs with distinct functions. Like all RNase III enzymes, Dicer leaves 2-nt 3′-overhangs and 5′ phosphate groups. Some organisms, including mammals and nematodes, have only a single Dicer enzyme that functions in the biogenesis of both miRNAs and siRNAs whereas other organisms divide the labor among multiple Dicer proteins. For example, two distinct enzymes are involved in parallel pathways for small regulatory RNA biogenesis in Drosophila. Dicer-2 processes long-double-stranded precursors and generates siRNAs, while Dicer-1 processes pre-miRNAs into mature miRNAs.
早期 Hannon、Zamore、Tuschl 及其同事的体外研究表明，RNAi 活性蛋白复合物包含与靶向 mRNA 同源的 25 核苷酸小 RNA。随后不久，几个实验室利用遗传和生化方法鉴定出 RNAi 通路中的酶和辅助因子。这些研究揭示双链 RNA 被 Dicer (RNase III 家族成员) 酶切割，生成 21-23 核苷酸的较短 RNA 双链 (图 1)。Dicer 是真核生物中保守的蛋白，可以具有多个功能不同的同源物。与所有 RNase III 酶一样，Dicer 留下的产物具有 2 个核苷酸的 3' 末端突出和 5' 磷酸酯基团。一些生物体，例如哺乳动物和线虫，只有一个 Dicer 酶，其在 microRNA 和 siRNA 的生物合成中发挥作用，而其他生物体则将这项工作分配给多个 Dicer 蛋白。例如，果蝇中存在两个独立的酶参与小调节 RNA 生物合成的平行途径。Dicer-2 处理长双链前体分子并产生 siRNA，而 Dicer-1 则将 pre-miRNA 加工成成熟的 miRNA。
Fig. 1 Schematic representation of gene silencing by double-stranded RNAs. The RNAi pathway is experimentally activated by introducing double-stranded RNA (dsRNA) to silence specific genes. Long dsRNA is cut into 19–21 bp siRNA fragments by Dicer and then into a multi-protein complex termed RNA-induced silencing complex (RISC) where the sense strand with high 5′-end stability is eliminated by the nuclease argonaute 2 (Ago2), resulting in strand separation. Synthetic siRNAs are directly loaded into the Ago2 RISC complex. Subsequent to strand separation, the sense strand guides the RISC to recognize and cleave complementary mRNA sequences. Gene silencing is a result of nucleolytic degradation of the targeted mRNA by Argonaute 2, an RNase H enzyme. Cleaved mRNA molecules are rapidly degraded by cellular nucleases. Following dissociation, the RISC is able to recycle and cleave additional mRNA molecules. Unlike chemically made siRNAs, hairpin RNAs (hsiRNAs) produced from plasmid vectors in cell nucleus are processed by Dicer in the cytoplasm before entering the RNAi pathway.
双链 RNA 基因沉默的示意图。通过向细胞中引入双链 RNA(dsRNA)可以实验性激活 RNAi 途径,从而沉默特定基因的表达。长双链 RNA 首先被 Dicer 酶剪切成 19-21 个碱基对的 siRNA 片段,然后被装载进一个称为 RNA 诱导沉默复合物(RISC)的多蛋白复合物中。在 RISC 复合物中,5'端更稳定的正义链被 Argonaute 2 核酶(Ago2)降解,导致双链分离。人工合成的 siRNA 则可以直接装载进 Ago2 RISC 复合物中。链分离后,RISC 复合物中剩余的反义链就能够指导 RISC 识别和剪切与之完全互补的靶标 mRNA 序列。基因表达被沉默的根本原因是靶标 mRNA 被 Argonaute 2(一种 RNase H 酶)降解。被剪切的 mRNA 片段随后会迅速被细胞核酸酶彻底降解。经过一轮作用后,RISC 复合物能够解离并循环利用,继续剪切其他靶标 mRNA。与化学合成的 siRNA 不同,从质粒载体在细胞核中产生的发卡 RNA(hsiRNA)在进入 RNAi 途径之前,需要先在细胞质中被 Dicer 酶加工处理。
In the next step, the siRNA duplexes are loaded onto an Argonaute protein , which along with Dicer and the trans-activation response RNA binding protein (TRBP) form the effector RNA-induced silencing complex (RISC). Subsequent to strand separation based on asymmetric unwinding, the antisense strand guides the activated RISC to the complementary site in the target mRNA, resulting in mRNA cleavage (Fig.1). In mammals, the catalytic activity of RISC is mediated by Ago2 protein. This slicer activity of the Ago-2 is very precise and cleaves in a position 10 and 11, counting from the 5′end, generating products with a 5′-monophosphate and a 3′-hydroxyl termini. Once the target mRNA is degraded by cellular nucleases, the RISC complex is recycled and free to cleave additional targets. Eukaryotic Argonaute protein consists of three conserved domains: PAZ, MID, and PWI. The PAZ domain binds the 3′end of the single-stranded RNA. The Mid domain folds into a binding pocked that anchors the 5′ phosphate of the small RNA. Therefore, it is a strict requirement for the siRNAs to be 5′ phosphorylated to enter into the RISC pathway, and siRNAs that lack a 5′ phosphate are rapidly phosphorylated by an endogenous kinase prior to loading into the RISC complex. Analysis of the crystal structure of a siRNA guide strand associated with an Ago2 PIWI domain identified a seed sequence (nucleotides 2–8) that directs target mRNA recognition by RISC. It should be noted that the interactions between the small RNA and Ago2 protein are mediated by the sugar-phosphate backbone; as a result, the nucleotides of the guide RNA are free to base-pair with target mRNA. In humans, there are four Argonaute proteins and only Ago2 is catalytically active. In flies and humans, the passenger strand cleaved by Ago2 is removed from the RISC-loading complex by the endonuclease C3PO, known as translin. Although the mechanisms of gene silencing by dsRNAs are well studied, additional factors may contribute to strand selection by RISC and cleavage activity. Moreover, competition of coding and non-coding RNAs for Argonaute binding can influence gene silencing.
下一步，siRNA 双链会加载到 Argonaute 蛋白上，Argonaute 蛋白与 Dicer 和转录激活应答 RNA 结合蛋白 (TRBP) 共同组成效应 RNA 诱导沉默复合物 (RISC)。经过不对称解旋导致链分离后，反义链指导激活的 RISC 复合物找到靶向 mRNA 上的互补序列，并切割 mRNA (图 1)。在哺乳动物中，RISC 的催化活性由 Ago2 蛋白介导。Ago2 具有高度精确的切割活性，可在距离 mRNA 5' 末端 10-11 个核苷酸的位置切割 mRNA，产生具有 5' 磷酸单酯和 3' 羟基末端的片段。一旦目标 mRNA 被细胞核酸酶降解，RISC 复合物就会被回收利用，继续切割其他的靶标。真核生物的 Argonaute 蛋白包含三个保守结构域：PAZ、MID 和 PWI。PAZ 结构域结合单链 RNA 的 3' 末端。MID 结构域折叠成一个结合口袋，锚定小 RNA 的 5' 磷酸基团。因此，siRNA 必须 5' 磷酸化才能进入 RISC 途径，缺乏 5' 磷酸基团的 siRNA 在加载到 RISC 复合物之前会被内源性激酶快速磷酸化。通过分析与 Ago2 PIWI 结域相关的 siRNA 引导链的晶体结构，揭示了一个种子序列 (核苷酸 2-8)，该序列指导 RISC 识别靶向 mRNA。需要指出的是，小 RNA 和 Ago2 蛋白之间的相互作用由糖磷酸骨架介导，因此引导 RNA 的核苷酸可以自由与靶向 mRNA 碱基配对。人类有四种 Argonaute 蛋白，只有 Ago2 具有催化活性。在果蝇和人类中，Ago2 切下的乘客链会被一种称为转运素 (translin) 的核酸内切酶 C3PO 从 RISC 加载复合物中去除。尽管科学家们已经很好地研究了双链 RNA 介导基因沉默的机制，但其他因素也可能影响 RISC 的链选择和切割活性。此外，编码 RNA 和非编码 RNA 与 Argonaute 结合的竞争会影响基因沉默。
