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RNA干扰研究现状和主要过程


原理、产生背景、发展历史、现状、发展趋势、理论或实践意义 May 20, 2002

Post-transcriptional gene silencing (PTGS), which was initially considered a bizarre phenomenon limited to petunias and a few other plant species, is now one of the hottest topics in molecular biology (1). In the last few years, it has become clear that PTGS occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. Perhaps most exciting, however, is the emerging use of PTGS and, in particular, RNA interference (RNAi) — PTGS initiated by the introduction of double-stranded RNA (dsRNA) — as a tool to knock out expression of specific genes in a variety of organisms (reviewed in 1-3). How was RNAi discovered? How does it work? Perhaps more importantly, how can it be harnessed for functional genomics experiments? This article will briefly answer these questions and provide you with resources to find in depth information on PTGS and RNAi research. More than a decade ago, a surprising observation was made in petunias. While trying to deepen the purple color of these flowers, Rich Jorgensen and colleagues introduced a pigment-producing gene under the control of a powerful promoter. Instead of the expected deep purple color, many of the flowers appeared variegated or even white. Jorgensen named the observed phenomenon "cosuppression", since the expression of both the introduced gene and the homologous endogenous gene was suppressed (1-5). First thought to be a quirk of petunias, cosuppression has since been found to occur in many species of plants. It has also been observed in fungi, and has been particularly well characterized in Neurospora crassa, where it is known as "quelling" (1-3). But what causes this gene silencing effect? Although transgene-induced silencing in some plants appears to involve gene-specific methylation (transcriptional gene silencing, or TGS), in others silencing occurs at the post-transcriptional level (post-transcriptional gene silencing, or PTGS). Nuclear run-on experiments in the latter case show that the homologous transcript is made, but that it is rapidly degraded in the cytoplasm and does not accumulate (1, 3, 6). Introduction of transgenes can trigger PTGS, however silencing can also be induced by the introduction of certain viruses (2, 3). Once triggered, PTGS is mediated by a diffusible,

trans-acting molecule. This was first demonstrated in Neurospora, when Cogoni and colleagues showed that gene silencing could be transferred between nuclei in heterokaryotic strains (1, 7). It was later confirmed in plants when Palauqui and colleagues induced PTGS in a host plant by grafting a silenced, transgene-containing source plant to an unsilenced host (8). From work done in nematodes and flies, we now know that the trans-acting factor responsible for PTGS in plants is dsRNA (1-3). RNAi Is Discovered in Nematodes The first evidence that dsRNA could lead to gene silencing came from work in the nematode Caenorhabditis elegans. Seven years ago, researchers Guo and Kemphues were attempting to use antisense RNA to shut down expression of the par-1 gene in order to assess its function. As expected, injection of the antisense RNA disrupted expression of

par-1, but quizzically, injection of the sense-strand control did too (9).
This result was a puzzle until three years later. It was then that Fire and Mello first injected dsRNA — a mixture of both sense and antisense strands — into C. elegans (10). This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Indeed, injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous gene's expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in its first generation offspring (10). The potency of RNAi inspired Fire and Timmons to try feeding nematodes bacteria that had been engineered to express dsRNA homologous to the C. elegans unc-22 gene. Surprisingly, these worms developed an unc-22 null-like phenotype (11-13). Further work showed that soaking worms in dsRNA was also able to induce silencing (14). These strategies, whereby large numbers of nematodes are exposed to dsRNA, have enabled large-scale screens to select for RNAi-defective C. elegans mutants and have led to large numbers of gene knockout studies within this organism (15-18). RNAi in Drosophila RNAi has also been observed in Drosophila. Although a strategy in which yeast were engineered to produce dsRNA and then fed to fruit flies failed to work, microinjecting

Drosophila embryos with dsRNA does effect silencing (2). Silencing can also be induced by
"shooting" dsRNA into Drosophila embryos with a "gene gun" or by engineering flies to carry DNA containing an inverted repeat of the gene to be silenced. Over the last few years, these RNAi strategies have been used as reverse genetics tools in Drosophila organisms, embryo lysates, and cells to characterize various loss-of-function phenotypes (2, 19-23).

So how does injection of dsRNA lead to gene silencing? Many research groups have diligently worked over the last few years to answer this important question. A key finding by Baulcombe and Hamilton provided the first clue. They identified RNAs of ~25 nucleotides in plants undergoing cosuppression that were absent in non-silenced plants. These RNAs were complementary to both the sense and antisense strands of the gene being silenced (24). Further work in Drosophila — using embryo lysates and an in vitro system derived from S2 cells — shed more light on the subject (3, 25, 26). In one notable series of experiments, Zamore and colleagues found that dsRNA added to Drosophila embryo lysates was processed to 21-23 nucleotide species. They also found that the homologous endogenous mRNA was cleaved only in the region corresponding to the introduced dsRNA and that cleavage occurred at 21-23 nucleotide intervals (26). Rapidly, the mechanism of RNAi was becoming clear. Current Models of the RNAi Mechanism Both biochemical and genetic approaches (see "The Genes and Enzymes Involved in PTGS and RNAi" below for a discussion of genetic approaches used to undersand RNAi) have led to the current models of the RNAi mechanism. In these models, RNAi includes both initiation and effector steps (27, see also a Flash animation of "How Does RNAi Work?", from reference 3). In the initiation step, input dsRNA is digested into 21-23 nucleotide small interfering RNAs (siRNAs), which have also been called "guide RNAs" (reviewed in 3, 18, 27). Evidence indicates that siRNAs are produced when the enzyme Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, processively cleaves dsRNA (introduced directly or via a transgene or virus) in an ATP-dependent, processive manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3' overhangs (27, 28). In the effector step, the siRNA duplexes bind to a nuclease complex to form what is known as the RNA-induced silencing complex, or RISC. An ATP-depending unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA ~12 nucleotides from the 3' terminus of the siRNA (3, 18, 27, 29). Although the mechanism of cleavage is at this date unclear, research indicates that each RISC contains a single siRNA and an RNase that appears to be distinct from Dicer (27).

Because of the remarkable potency of RNAi in some organisms, an amplification step within the RNAi pathway has also been proposed. Amplification could occur by copying of the input dsRNAs, which would generate more siRNAs, or by replication of the siRNAs themselves (see "Possible Role for RNA-dependent RNA Polymerase" below). Alternatively or in addition, amplification could be effected by multiple turnover events of the RISC (3, 18, 27). Possible Role for RNA-dependent RNA Polymerase Genetic screens in Neurospora, C. elegans, and Arabidopsis have identified several genes that appear to be crucial for PTGS and RNAi. Several of these, including Neurospora qde-1,

Arabidopsis SDE-1/SGS-2 and C. elegans ego-1, appear to encode RNA-dependent RNA
polymerases (RdRPs). At first glance, it might be assumed that this is proof that an RdRP activity is required for RNAi. Certainly the existence of an RdRP might explain the remarkable efficiency of dsRNA-induced silencing if it amplifed either the dsRNA prior to cleavage or the siRNAs directly. But mutants of these genes have varying phenotypes, which makes the role of RdRP in RNAi difficult to discern (1, 3, 17, 18). In C. elegans ego-1 mutants ("ego" stands for "enhancer of glp-1"), RNAi functions normally in somatic cells, but is defective in germline cells where ego-1 is primarily expressed. In Arabidopsis SDE-1/SGS-2 mutants ("SGS" stands for suppressor of gene silencing), siRNAs are produced when dsRNA is introduced via an endogenously replicating RNA virus, but not when introduced by a transgene. It has been proposed that perhaps the viral RdRP is substituting for the Arabidopsis enzyme in these mutants (1, 3, 17, 18). Although no homolog of an RdRP has been found in flies or humans, an RdRP activity has recently been reported in Drosophila embryo lysates (30). One model of amplification, termed the "random degradative PCR" model, suggests that an RdRP uses the guide strand of an siRNA as a primer for the target mRNA, generating a dsRNA substrate for Dicer and thus more siRNAs (27, 30). Evidence supporting this model has been found in worms, whereas experimental results refuting the model have been obtained from

Drosophila embryo lysates (26, 27).
RNAi Initiators Two C. elegans genes, rde-1 and rde-4 ("rde" stands for "RNAi deficient"), are believed to be involved in the initiation step of RNAi. Mutants of these genes produce animals that are resistant to silencing by injection of dsRNA, but silencing can be effected in these animals by the transmission of siRNA from heterozygous parents that are not silencing deficient. The C. elegans rde-1 gene is a member of a large family of genes and is homologous to the

Neurospora qde-2 ("qde" stands for "quelling deficient") and the Arabidopsis AGO1 genes
("AGO" stands for "argonaute"; AGO1 was previously identified to be involved in

Arabidopsis development). Although the function of these genes in PTGS is unclear, a
mammalian member of the RDE-1 family has been identified as a translation initiation factor. Interestingly, Arabidopsis mutants of AGO1, which are defective for cosuppression, also exhibit defects in leaf development. Thus some processes or enzymes involved in PTGS may also be involved in development (1, 3, 17, 18). RNAi Effectors Important genes for the effector step of PTGS include the C. elegans rde-2 and mut-7 genes. These genes were initially identified from heterozygous mutant worms that were unable to transmit RNAi to their homozygous offspring (16). Worms with mutated rde-2 or

mut-7 genes exhibit defective RNAi, but interestingly, they also demonstrate increased
levels of transposon activity. Thus, silencing of transposons appears to occur by a mechanism related to RNAi and PTGS. Although the rde-2 gene product has not yet been identified, the mut-7 gene encodes a protein with homology to the nuclease domains of RNase D and a protein implicated in Werner syndrome (a rapid aging disease) in humans (1, 3, 17, 18, 31). Perhaps this protein is a candidate for the nuclease activity required for target RNA degradation. PTGS Has Ancient Roots Discoveries from both genetic and biochemical approaches point to the fact that PTGS has deep evolutionary roots. Proposals have been put forth that PTGS evolved as a defense mechanism against transposons or RNA viruses, perhaps before plants and animals diverged (1, 3, 17, 18). Interestingly, it was noted by many researchers that disruption of genes required for RNAi often causes severe developmental defects. This observation suggested a link between RNAi and at least one developmental pathway. A group of small RNA molecules, known as small temporal RNAs (stRNAs), regulates C.

elegans developmental timing through translational repression of target transcripts.
Research indicates that the C. elegans lin-4 and let-7 stRNAs are generated from 70-nt transcripts following the folding of these longer transcripts into a stem-loop structure. The folded RNA molecules are cleaved to produce 22-nt stRNAs by the enzyme Dicer (called DCR-1 in C. elegans). Thus Dicer generates both siRNAs and stRNAs, and represents an intersection point for the RNAi and stRNA pathways (32-34).

Recently, nearly 100 additional ~22 nt RNA molecules, termed microRNAs (miRNAs), were identified in Drosophila, C. elegans, and HeLa cells (35-38). Much like lin-4 and let-7, these miRNAs are formed from precursor RNA molecules that fold into a stem-loop secondary structure. The newly discovered ~22 nt miRNAs are believed to play a role in regulation of gene expression, and at least two of them are known to require Dicer for their production (37). It appears that the use of small RNAs for both gene regulation and RNAi is a common theme throughout evolution. Non-specific Gene Silencing by Long dsRNAs While the natural presence of RNAi had been observed in a variety of organisms (plants, protozoa, insects, and nematodes), evidence for the existence of RNAi in mammalian cells took longer to establish. Transfection of long dsRNA molecules (>30 nt) into most mammalian cells causes nonspecific suppression of gene expression, as opposed to the gene-specific suppression seen in other organisms. This suppression has been attributed to an antiviral response, which takes place through one of two pathways. In one pathway, long dsRNAs activate a protein kinase, PKR. Activated PKR, in turn phoshorylates and inactivates the translation initiation factor, eIF2a, leading to repression of translation. (39) In the other pathway, long dsRNAs activate RNase L, which leads to nonspecific RNA degradation (40). A number of groups have shown that the dsRNA-induced antiviral response is absent from mouse embryonic stem (ES) cells and at least one cell line of embryonic origin. (41, 42) It is therefore possible to use long dsRNAs to silence specific genes in these specific mammalian cells. However, the antiviral response precludes the use of long dsRNAs to induce RNAi in most other mammalian cell types. siRNAs Bypass the Antiviral Response Interestingly, dsRNAs less than 30 nt in length do not activate the PKR kinase pathway. This observation, as well as knowledge that long dsRNAs are cleaved to form siRNAs in worms and flies and that siRNAs can induce RNAi in Drosophila embryo lysates, prompted researchers to test whether introduction of siRNAs could induce gene-specific silencing in mammalian cells (43). Indeed, siRNAs introduced by transient transfection were found to effectively induce RNAi in mammalian cultured cells in a sequence-specific manner. The effectiveness of siRNAs varies — the most potent siRNAs result in >90% reduction in target RNA and protein levels (44-46). The most effective siRNAs turn out to be 21 nt dsRNAs with 2 nt 3' overhangs. Sequence specificity of siRNA is very stringent, as single base pair mismatches between the siRNA and its target mRNA dramatically reduce

silencing (44, 47). Unfortunately, not all siRNAs with these characteristics are effective. The reasons for this are unclear but may be a result of positional effects (46, 48, 49). For current recommendations on designing siRNAs, see "siRNA Design". Although the history and mechanism of RNAi and PTGS are fascinating, many researchers are most excited about RNAi's potential use as a functional genomics tool. Already RNAi has been used to ascertain the function of many genes in Drosophila, C. elegans, and several species of plants. With the knowledge that RNAi can be induced in mammalian cells by the transfection of siRNAs, many more researchers are beginning to use RNAi as a tool in human, mouse and other mammalian cell culture systems. In early experiments with mammalian cells, the siRNAs were synthesized chemically (Ambion is one of several companies that offer custom siRNA synthesis). Recently, Ambion introduced a kit (the Silencer? siRNA Construction Kit) to produce siRNAs by in vitro transcription, which is a less expensive alternative to chemical synthesis, particularly when multiple different siRNAs need to be synthesized. Once made, the siRNAs are introduced into cells via transient transfection. Due to differences in efficacy, most researchers will synthesize 3–4 siRNAs to a target gene and perform pilot experiments to determine the most effective one. Transient silencing of more than 90% has been observed with this type of approach (44-46, 48, 49). So far, injection and transfection of dsRNA into cells and organisms have been the main method of delivery of siRNA. And while the silencing effect lasts for several days and does appear to be transferred to daughter cells, it does eventually diminish. Recently, however, a number of groups have developed expression vectors to continually express siRNAs in transiently and stably transfected mammalian cells (50-56). Some of these vectors have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (50, 53, 54, 56). The vectors contain the shRNA sequence between a polymerase III (pol III) promoter and a 4-5 thymidine transcription termination site. The transcript is terminated at position 2 of the termination site (pol III transcripts naturally lack poly(A) tails) and then folds into a stem-loop structure with 3' UU-overhangs. The ends of the shRNAs are processed in vivo, converting the shRNAs into ~21 nt siRNA-like molecules, which in turn initiate RNAi (50). This latter finding correlates with recent experiments in C. elegans, Drosophila, plants and Trypanosomes, where RNAi has been induced by an RNA molecule that folds into a stem-loop structure (reviewed in 3).

Another siRNA expression vector developed by a different research group encodes the sense and antisense siRNA strands under control of separate pol III promoters (52). The siRNA strands from this vector, like the shRNAs of the other vectors, have 5 thymidine termination signals. Silencing efficacy by both types of expression vectors was comparable to that induced by transiently transfecting siRNA. The recent studies on RNAi have taken the research world by storm. The ability to quickly and easily create loss-of-function phenotypes has researchers rushing to learn as much as they can about RNAi and the characteristics of effective siRNAs. In the future, RNAi may even hold promise for development of gene-specific therapeutics. Much has been learned about this powerful technique, but additional information becomes available on an almost daily basis (see RNAi/index.html">The RNA Interference Resource to learn about the very latest RNAi research and tools). It is not an understatement to say that the field of functional genomics is being revolutionized by RNAi. Related Articles
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1995 年,康乃尔大学的 Su Guo 博士在试图阻断秀丽新小杆线虫(C. elegans)中的 par-1 基 因时,发现了一个意想不到的现象。她们本是利用反义 RNA 技术特异性地阻断上述基因的 表达,而同时在对照实验中给线虫注射正义 RNA(sense RNA)以期观察到基因表达的增强。 但得到的结果是二者都同样地切断了 par-1 基因的表达途径。 这是与传统上对反义 RNA 技术 的解释正好相反的。该研究小组一直没能给这个意外以合理解释。 直到 1998 年 2 月, 华盛顿卡耐基研究院的 Andrew Fire 和马萨诸塞大学医学院的 Craig Mello 才首次揭开这个悬疑之谜。通过大量艰苦的工作,他们证实,Su Guo 博士遇到的正义 RNA 抑制基因表达的现象,以及过去的反义 RNA 技术对基因表达的阻断,都是由于体外转录所 得 RNA 中污染了微量双链 RNA 而引起。 当他们将体外转录得到的单链 RNA 纯化后注射线虫 时发现,基因抑制效应变得十分微弱,而经过纯化的双链 RNA 却正好相反,能够高效特异 性阻断相应基因的表达。该小组将这一现象称为 RNA 干扰(RNA interference ,简称 RNAi) 。 在随后的短短一年中,RNAi 现象被广泛地发现于真菌、拟南芥、水螅、涡虫、锥虫、斑马 鱼等大多数真核生物中。这种存在揭示了 RNAi 很可能是出现于生命进化的早期阶段。随着 研究的不断深入,RNAi 的机制正在被逐步阐明,而同时作为功能基因组研究领域中的有力 工具,RNAi 也越来越为人们所重视。 RNAi 的作用机制 RNAi 作用机制 体外实验表明:RNAi 反应中,加入的 dsRNA 被切割为 21-23 核苷酸长的 RNA 片段,后者会 使目的 mRNA 被切割为 21-23 核苷酸长的片段。 从已经发生 RNAi 的果蝇 S2 细胞中, Hammond 等人部分纯化了一种核酸酶,该核酸酶具有序列特异性,它仅降解与引起 RNAi 的 dsRNA 具 有同源序列的 mRNA。 那么这种核酸酶是如何确定哪些 mRNA 该降解而哪些不该呢?由于在 纯化该核酸酶时,可以共分离出 21-23 核苷酸长的 dsRNA 片段,这暗示该核酸酶对 mRNA 的切割有可能是以这些片段作模板指导进行的。根据以上的实验结果,人们提出一种 RNAi 作用的简单模型。 dsRNA 导入细胞后, 当 被一种 dsRNA 特异的核酸内切酶识别, 切割成 21-23 核苷酸长的小片段,这些片段可与该核酸酶的 dsRNA 结合结构域结合,并且作为模板识别 目的 mRNA;识别之后,mRNA 与 dsRNA 的有义链发生链互换,原先 dsRNA 中的有义链被 mRNA 代替,从酶-dsRNA 复合物中释放出来,而 mRNA 则处于原先的有义链的位置。核酸 酶在同样位置对 mRNA 进行切割,这样又产生了 21-23 核苷酸长的 dsRNA 小片段,与核酸 酶形成复合物,继续对目的 mRNA 进行切割,从而使目的基因沉默,产生 RNAi 现象。通过 遗传分析的方法,目前已从线虫中已分离到 RDE-2,RDE-3 和 Mut-7 等 RNAi 相关的基因。 RNAi 技术在功能基因组中的应用 在功能基因组研究中, 需要对特定基因进行功能丧失或降低突变, 以确定其功能。 由于 RNAi

具有高度的序列专一性, 可以特异地使特定基因沉默, 获得功能丧失或降低突变, 因此 RNAi 可以作为一种强有力的研究工具,用于功能基因组的研究。将功能未知的基因的编码区(外 显子)或启动子区,以反向重复的方式由同一启动子控制表达。这样在转基因个体内转录出 的 RNA 可形成 dsRNA,产生 RNA 干涉,使目的基因沉默,从而进一步研究目的基因的功能, 这种技术即为 RNAi 技术。 根据所选用序列的不同, 可将其分为编码区 RNAi 和启动子区 RNAi 技术。 1.编码区 RNAi 技术 自 1998 年在线虫中发现 RNAi 现象以来,以基因编码区为靶序列的编码区 RNAi 技术已用于 线虫功能基因组的研究。 最初这种技术是通过注射或浸泡等方法直接导入到线虫的性腺或早 期胚胎中。这些方法虽然可以关闭目的基因的表达,产生突变表型,但这种表型变化却不能 遗传。这种早期的 RNAi 技术可以用于研究与胚胎发育有关的基因的功能,但由于细胞分裂 造成 dsRNA 的稀释,使得这种方法在研究成体的基因功能时有一定的局限性。为弥补早期 RNAi 技术的上述不足, Tavernarakis 等对 RNAi 技术进行了改进, 将目的基因的靶序列以反向 重复的方式,由热激启动子控制在转基因生物中表达。热激处理后,反向重复序列在细胞内 开始转录,其产物会形成具发夹环结构的 dsRNA,从而产生 RNAi,使目的基因沉默。这种 改进的 RNAi 技术与传统的 RNAi 技术相比,具有明显的优点:首先转基因可以遗传给后代, 有利于突变的分析;其次 dsRNA 可以被诱导产生,RNAi 能够在发育特定阶段出现,从而使 研究发育早期必需基因在发育晚期的功能成为可能;另外,当用细胞特异性启动子控制 dsRNA 的表达时,可以研究特定基因在不同器官中的功能。Kennerdell J. R.和 CarthewR. W. 用 GAL4/UAS 系统控制 dsRNA 在果蝇中的表达,实现了诱导性或细胞特异性控制 RNAi 的发 生。 随着应用 RNAi 技术研究线虫功能基因组工作的开展,研究人员对该技术在植物中应用的可 能性进行了探索。加州理工大学的 Chuang C. F.和 Megerowitz E. M.使用此技术研究了拟南芥 的 AG, CLV3, AP1, PAN 四个开花相关基因。 结果表明使用 RNAi 技术可以产生功能丧失或降低 突变体,其表型与以前通过其它方法鉴定的突变体类似。RNA 原位杂交表明,RNAi 突变体 的目的 mRNA 显著降低。该结果说明 RNAi 技术亦可以成为植物功能基因组研究中的有力工 具。 2.启动子区 RNAi 技术 M. F. Mett 等证明含有启动子区的 dsRNA 在植物体内同样被切割成 21-23 核苷酸长的片段, 这种 dsRNA 可使内源相应的 DNA 序列甲基化, 从而使启动子失去功能, 使其下游基因沉默。 由于多基因家族的各成员之间高度同源,因而使用编码区 RNAi 技术很难将各个成员区分开 来研究,而多基因家族内的启动子序列通常比编码区变化大,采用启动子区 RNAi 技术有望 将多基因家族的各个成员区分开来研究。 这样综合编码区 RNAi 技术和启动子区 RNAi 技术的 信息即可更全面地了解多基因家族地各成员的功能。 RNAi 现象存在的广泛性远远超过人们 的预期, 对此问题的深入研究结果将为进化的观点提供有力佐证。 而与其它几种进行功能丧 失或降低突变的技术相比,RNAi 技术具有明显的优点,它比反义 RNA 技术和同源共抑制更 有效,更容易产生功能丧失或降低突变。而且通过 与细胞特异性启动子及可诱导系统结合使用, 可以在发育的不同时期或不同器官中有选择地 进行,与 T-DNA 技术造成的功能永久性缺失相比,这是更受科学家偏爱的。我坚信,由于

科学家们的努力工作,一个崭新的 RNA 时代呼之欲出。 RNAi 的定义 目前对 RNAi (RNA interference)的定义有很多种, 不同的资料对其定义的侧重点 也不尽相同,如果将 RNAi 看作一种生物学现象,可以有以下定义:① RNAi 是由 dsRNA 介导的由特定酶参与的特异性基因沉默现象,它在转录水平、转录后水平和翻 译水平上阻断基因的表达。② RNAi 是有 dsRNA 参与指导的,以外源和内源 mRNA 为降解目标的转基因沉默现象。具有核苷酸序列特异性的自我防御机制,是一种当外 源基因导入或病毒入侵后,细胞中与转基因或入侵病毒 RNA 同源的基因发生共同基 因沉默的现象。 如果将其作为一门生物技术,则定义为:① RNAi 是指通过反义 RNA 与正链 RNA 形成双链 RNA 特异性地抑制靶基因的现象,它通过人为地引入与内源靶基因具 有相同序列的双链 RNA(有义 RNA 和反义 RNA) ,从而诱导内源靶基因的 mRNA 降 解 ,达 到 阻 止 基 因 表 达 的 目 的 。 ② RNAi 是 指 体 外 人 工 合 成 的 或 体 内 的 双 链 RNA (dsRNA)在细胞内特异性的将与之同源的 mRNA 降解成 21nt~23nt 的小片段, 使相应的基因沉默。 RNAi 是将与靶基因的 mRNA 同源互补的双链 RNA(dsRNA ) ③ 导入细胞,能特异性地降解该 mRNA ,从而产生相应的功能表型缺失, 属于转录后水平 的基因沉默(post - transcriptional gene silence , PTGS)。 各种不同定义虽然说法不同,但所描述事实是大体相同的,简单地可以说,RNAi 就是指由 RNA 介导的基因沉默现象。 最近由于 RNA 干扰(RNA interference,RNAi)的发现使反义领域的研究增多。 这种自然发生的现象最早是在秀丽线虫中发现的(1) ,是序列特异性地使转录后的基 因沉默的有力机制。由于最近两年在 RNAi 领域取得的进步,已经有许多这方面的综 述发表 (2-4) RNA 干扰是由长的双链 RNA 分子发动的, 。 该分子可以被 Dicer enzyme 加工成长度为 21-23 个核苷酸的 RNA(见图) 。RNaseIII 蛋白被认为是作为一个二聚 体发挥作用,它对双链 RNA 的两个链都进行切割,酶切的产物 3'末端互相重叠。然 后这种小的干扰 RNA 分子(small interfering RNAs,siRNAs)掺入 RNA 诱导的沉 默复合物(RNA-induced silencing complex,RISC) ,引导核酸酶降解靶 RNA。 这种保守的生化机制可用于研究多种模式生物的基因功能,但是它在哺乳动物细 胞中的应用受到阻碍,因为长的双链 RNA 分子会引起干扰素应答。因此 Tuschi 及其 同事表明长度为 21nt 的 siRNA 可以特异性的抑制哺乳动物细胞基因表达是一个革命 性的突破(5) 。这个发现激发了大量利用 RNAi 技术对哺乳动物细胞的研究,因为与 传统的反义技术比,RNAi 的性能明显较高。 有趣的是,除了短双链 RNA,短发夹 RNA(short hairpin RNA,shRNA) ,比如 茎环结构在细胞内经过加工后也可以变成 siRNA,从而产生 RNA 干扰(6、7) 。这使 得构建表达干扰 RNA 的载体, 从而使哺乳动物细胞内基因表达长期沉默成为可能 (4、 8) 。shRNA 可以利用 RNA 聚核酶 III 启动子转录,在正常情况下,该启动子是控制 小核 RNA(small nuclear RNA,snRNA)U6(6、7、9、10)或者 RNaseP 的组分 H1 RNA(11)转录的。另外一种办法是两段短 RNA 分子分别用 U6 启动子转录出来

(6、12、13) 。载体介导的 siRNA 表达使对功能缺失(loss-of-function)表型进行 长期分析成为可能。在稳定转染的细胞内,两个月后仍可观察到沉默现象(11) 。 另外一种延长 siRNA 抑制基因表达时间的方法是对化学合成的 RNA 进行核苷酸 修饰。尽管未经修饰的短双链 RNA 在细胞培养物或者体内的稳定性出乎意料的高, 然而有些情况下,需要对 siRNA 的稳定性进行进一步提高。因此,可以在两条链的 末端都引入经过修饰的核苷(14) 。一个 5'端为两个 2'-O-甲基 RNA、3'端为 4 个甲基 化核苷的 siRNA 与序列相同但是未经修饰的 siRNA 比活性相同,但是在细胞培养物 中引起的基因沉默现象的时间延长。然而,增多 siRNA 中的甲基化核苷,或者在核 苷中引入体积较大的烯丙基将导致 siRNA 活性下降。 RNA 干扰在哺乳动物体内的第一个研究是利用快速注射大量生理溶液的方法将 一个编码 shRNA 的质粒注入老鼠的尾静脉(15、16) 。在大多数器官中,报道基因 (编码于共转染质粒或者转基因小鼠上)的表达可以被有效地抑制。另外,Fas 基因 被作为肝损伤治疗相关的内源靶标进行了 RNA 干扰实验(17) 。注射 siRNA 之后, 小鼠肝细胞中的 Fas mRNA 和蛋白水平下降了 10 天。 Fas 基因沉默可以保护小鼠 把 免遭由注射竞争性 Fas 特异抗体引起的爆发性肝炎,82%用 siRNA 处理的小鼠活过 了 10 天观察期,而所有的对照小鼠在 3 天之内死亡。 上述研究中采用的高压导入技术是一种粗暴的方法,不适于治疗用。因此,标准 的基因治疗所采用的方法被用于 RNA 干扰。 一个反转录病毒载体被用于导入 siRNA, 以抑制人类胰腺肿瘤细胞中的癌基因 K-ras 等位基因(18) 。负调控癌细胞中 K-ras 基因的表达使得它们在注入无胸腺的裸鼠皮下之后不再具有形成肿瘤的能力。这项研 究还表明 siRNA 的高度特异性,因为只有癌基因 K-ras 被沉默,而与之只有 1 个碱 基对差异的野生型等位基因并没有被沉默。另外,当在纹状区注射表达 siRNA 的腺 病毒之后,转基因小鼠大脑中 GFP 基因的表达可以被抑制(19) 。β-葡萄糖醛酸苷 酶 (b-glucoronidase) 的活性可以通过在小鼠尾静脉注射重组腺病毒抑制。 有趣的是, 具有 CMV 启动子和最小的 polyA 尾的 RNA 聚合酶 II 表达元件被用于这个实验,为 设计组织特异性或者可诱导的 siRNA 载体打开了大门。 总的来说,siRNA 的第一个体内实验已经进行,其他有重要意义的基因有望于很 快作为靶标开展研究。至今为止的研究没有观察到任何应用 siRNA 引起的毒性作用, 但是在治疗人类疾病的临床试验开始之前仍需小心,以排除长期使用 RNA 干扰引起 的严重副作用。因为用 siRNA 使基因表达沉默与传统的反义技术相似,研究者将从 十多年来反义技术研究的教训中获益,比如需要使用合适的对照以证明基因表达的敲 除是特异性的,以及对免疫系统可能引起的意外影响进行详细分析。 总结 经过长期盛衰沉浮,反义技术近年来得到越来越多的注意。对能够提高靶表亲和 性和生物稳定性、 降低毒性的修饰核苷的研究取得了重要进展。 由于大多数新的 DNA 类似物不能激活 RNaseH,对反义寡核苷酸的设计需要考虑靶 mRNA 是否需要保留, 例如,是改变剪接方式,还是降解靶 mRNA(这种情况下应该使用 gapmer 技术) 。 可以通过有系统 的修饰 天然核酶或者通 过体外 选择技术获得具 有高催 化活性的稳定 核酶。一些反义寡核苷酸和核酶已经进入临床试验研究,一个反义药物已经在 1998

年获得批准。一个重要的突破是发现短的双链 RNA 分子可用于哺乳动物细胞中特异 性沉默基因表达。这个方法与传统的反义技术比效率明显更高,并且一些体内实验的 数据已经发表。因此,反义技术有望广泛应用于对未知功能基因的研究、药物靶标的 确认和治疗。


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