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Although researchers demonstrated RNAi functionality using exogenous dsRNAs, RNAi is also induced by the endogenously present single-stranded hairpin microRNAs (miRNAs) (miRNAs) in cells. To date, miRNAs are the most abundant species of noncoding RNAs in mammalian cells, and they have fundamental roles in regulating gene expression at the posttranscriptional level. In some cases, RNAi can also confer resistance to virus or other pathogen infection. Notably, the first miRNAs were discovered through forward genetic screens in worms. Indeed, Ambros and colleagues described in 1993 a new mechanism of gene regulation in C. elegans through small non-protein-coding RNA lin-4, which was found to have complementary sequence to the 3′ untranslated region of lin-14 mRNA. In both plants and animals, cloning was the initial strategy of large-scale miRNA discovery. Additionally, several groups developed bioinformatics approaches for miRNA identification. According the miRBase database (release 22.1, http://www.mirbase.org ( http://www.mirbase.org )), there is currently 2656 human mature miRNA sequences that have the capacity to regulate more than two-thirds of protein-coding genes in humans and are involved in every cellular process known so far.
虽然研究人员最初是利用外源性双链 RNA 证实了 RNAi 的功能,但 RNAi 也可以被细胞内源性存在的单链发卡 microRNA(miRNA)诱导。至今,miRNA 是哺乳动物细胞中种类最丰富的非编码 RNA,在调控转录后基因表达水平上发挥着关键作用。在某些情况下,RNAi 还能赋予细胞对病毒或其他病原体感染的抗性。值得一提的是,首批 miRNA 是通过对线虫进行正向遗传学筛选发现的。1993 年,Ambros 等人描述了一种在线虫中存在的新型基因调控机制,即小的非编码 RNA lin-4 能与 lin-14 mRNA 的 3'非翻译区形成碱基互补配对。无论是在植物还是动物中,克隆技术最初都是大规模发现 miRNA 的主要策略。此外,一些研究小组也发展了生物信息学方法来鉴定 miRNA。根据 miRBase 数据库(22.1 版本,http://www.mirbase.org)的信息,目前已经有 2656 种人源成熟 miRNA 序列被鉴定出来,这些 miRNA 有能力调控人体超过三分之二的蛋白编码基因,并参与已知的几乎所有细胞过程。
In contrast to siRNAs, most miRNA genes are transcribed in the nucleus by RNA polymerase II, and the primary transcripts known as primary miRNAs (pri-miRNAs) are capped and polyadenylated. Before their export to the cytoplasm for further processing and silencing, nuclear pri-miRNAs must be recognized and cleaved (Fig. 2). These respective tasks are carried out by the proteins Drosha and DGCR8 (known as Pasha in invertebrates), which together constitute the microprocessor complex. As with Dicer, Drosha proteins belong to the RNase III family. The endonucleolytic activity results in the cleavage of the stem structure of pri-miRNAs and the release of the pre-miRNAs, which have a hairpin-like structure. Thereafter, Drosha-generated pre-miRNAs are exported from the nucleus to the cytoplasm by the nuclear export protein Exportin-5, which binds miRNA hairpin duplex along with GTP-bound form of the cofactor Ran, and releases it in the cytoplasm after hydrolysis of GTP. In the cytoplasm, pre-miRNAs are cleaved further by Dicer to generate 22-nt miRNA duplexes containing mismatches and having a two nucleotides 3′ overhang. Subsequently, the miRNA duplex assembles with Ago2 protein to form the effector RISC complex after which the mature miRNA strand is selected (Fig.2). Depending on the homology to mRNAs, miRNA can lead either to mRNA cleavage (complete homology) or to repression of translation (partial homology). In flies and mammals few miRNAs are reported to lead to cleavage of their targets, whereas in plants most miRNAs exhibit complete homology with their corresponding target mRNA genes , resulting in target cleavage. Plant miRNA genes are generally not located within protein-coding genes but comprise their own RNA polymerase II-dependent transcriptional units. This contrasts with animal miRNAs, which sometimes appear to be processed from introns of protein-coding genes. In plants, many predicted miRNA targets encode regulatory proteins, suggesting that plant miRNAs are master regulators. Notably, miRNAs and siRNAs have much in common. Both are 20–24-nucleotides long and processed from longer RNA precursors by Dicer-like ribonucleases, and both are incorporated into ribonucleoprotein silencing complexes in which the small RNAs, through their base-pairing potential, guide target gene repression. However, the fundamental difference between the two small RNA classes is the nature of their precursors; siRNAs are processed from long, double-stranded RNAs, whereas miRNAs are processed from single RNA molecules that include imperfect stem-loop secondary structures as illustrated in Fig.2.
与 siRNA 不同，大多数 miRNA 基因由 RNA 聚合酶 II 在细胞核内转录，产生的初级转录物称为原 miRNA (pri-miRNA)，具有帽子结构和 polyA 尾巴。这些原 miRNA 在被运出细胞核进行进一步加工和介导沉默之前，必须经过识别和切割 (图 2)。这项工作分别由蛋白质 Drosha 和 DGCR8 (无脊椎动物中称为 Pasha) 完成，它们共同组成微处理器复合体。与 Dicer 一样，Drosha 蛋白也属于 RNase III 家族。Drosha 的核酸内切酶活性可以切割原 miRNA 的茎环结构，释放具有发卡结构的 pre-miRNA。接下来，由 Drosha 产生的 pre-miRNA 与 GTP 结合形式的辅助因子 Ran 结合，并由核输出蛋白 Exportin-5 运出细胞核到细胞质中。Exportin-5 在水解 GTP 后将 miRNA 发卡双链释放到细胞质。在细胞质中，pre-miRNA 被 Dicer 进一步切割成包含错配并且具有两个核苷酸 3' 末端 overhang 的 22 核苷酸 miRNA 双链。随后，miRNA 双链与 Ago2 蛋白结合形成效应 RISC 复合物，然后选择成熟的 miRNA 链 (图 2)。miRNA 可以根据与 mRNA 的同源性导致 mRNA 切割 (完全同源) 或翻译抑制 (部分同源)。据报道，在果蝇和哺乳动物中，只有少数 miRNA 会导致靶标 mRNA 的切割，而在植物中，大多数 miRNA 与其对应的靶标 mRNA 具有完全同源性，导致靶标切割。植物 miRNA 基因通常不位于蛋白质编码基因内，而是具有依赖 RNA 聚合酶 II 的独立转录单元。这与动物 miRNA 形成对比，动物 miRNA 有时似乎是从蛋白质编码基因的内含子中加工而来。在植物中，许多预测的 miRNA 靶标编码调控蛋白，这表明植物 miRNA 是主要的调控因子。值得注意的是，miRNA 和 siRNA 有许多共同点。两者都长度为 20-24 个核苷酸，并由 Dicer 样核酸酶从较长的 RNA 前体分子加工而来，并且都结合到核糖蛋白沉默复合物中，通过碱基配对指导靶基因的抑制。然而，这两种小 RNA 类之间最主要的区别在于其前体的性质；siRNA 由长的双链 RNA 加工而来，而 miRNA 则由包含不完美茎环二级结构的单 RNA 分子加工而来，如图 2 所示。
Fig. 2 Schematic representation of miRNA silencing pathway. miRNAs are encoded in the genome and are transcribed to yield primary transcripts (pri-miRNAs), which are processed by Drosha with the aid of its partner DGCR8 protein (Pasha) to generate short precursor miRNAs (pre-miRNAs). These precursor miRNAs are then exported from the nucleus to the cytoplasm, where they are further processed by Dicer to yield miRNA duplexes that were unwound to single-stranded mature miRNA and then loaded into the miRNA-induced silencing complex (miRISC). Unwinding of the complex seems to occur so rapidly after duplex formation due to the Ago2 presence in the complex with Dicer and TRBP. The loaded strand RNA guides the RISC complex to recognize target mRNA sequences. A perfect homology allows Ago-cleavage of mRNA, whereas central mismatches promote repression of mRNA translation.miRNA 沉默途径的示意图。miRNA 由基因编码，转录成初级转录物 (pri-miRNA)。Drosha 蛋白在其辅助蛋白 DGCR8 (Pasha) 的帮助下加工 pri-miRNA，生成短的前体 miRNA (pre-miRNA)。这些前体 miRNA 随后从细胞核转运到细胞质，在那里被 Dicer 进一步加工成 miRNA 双链，然后解旋成单链成熟 miRNA，最后加载到 miRNA 诱导的沉默复合物 (miRISC) 中。由于 Dicer 和 TRBP 与 Ago2 共同存在于复合物中，因此复合物似乎在双链形成后立即解旋。加载的 RNA 链指导 miRISC 复合物识别靶向 mRNA 序列。如果 miRNA 与靶标 mRNA 完全同源,Ago2 就会剪切靶标 mRNA;而如果存在中心错配,则会抑制靶标 mRNA 的翻译过程。
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The Piwi-interacting RNAs (piRNAs) are the most recently discovered class of small non-coding RNAs, and they bind to the PIWI subclade of Argonaute proteins. One well-characterized Piwi/piRNA function is the silencing of retrotransposons, both at the posttranscriptional and epigenetic levels, as well as of other genetic elements in germlines, particularly those during spermatogenesis. Fly piRNAs were initially termed repeat-associated small interfering RNAs (rasiRNAs) because they were found to map to transposons and repetitive elements. Unlike rasiRNAs in flies, the majority of piRNAs in mammals arise from un-annotated regions of the genome devoid of transposons. Currently there are about 23,500 piRNAs identified in the human genome, a number that is quite comparable to that of protein-coding mRNAs (around 20,000), suggesting that piRNAs may have several important roles in gene regulation that remain to be investigated.
Piwi-相互作用 RNA(piRNA)是最新发现的一类小分子非编码 RNA，它们与 Argonaute 蛋白的 PIWI 亚家族成员结合。piRNA/Piwi 的一个重要功能是在转录后和表观遗传水平上沉默反转录转座子和其他遗传元件，尤其是在生殖细胞系中发生的精子发生过程。最初在果蝇中发现的 piRNA 被称为重复相关小干扰 RNA(rasiRNA)，因为它们被发现主要映射到转座子和重复序列上。与果蝇不同,哺乳动物中大部分 piRNA 来源于基因组中不含转座子的未注释区域。目前在人类基因组中已经鉴定出约 23,500 种 piRNA,这个数量与编码蛋白的 mRNA(约 20,000 个)相当，暗示 piRNA 可能在基因表达调控中发挥着一些尚待深入研究的重要功能。
Unlike miRNA and siRNA, piRNAs are typically longer, with a length of 26–31, originate from single-stranded RNA precursors, and do not require Dicer for maturation (Fig. 3). Most piRNAs are transcribed by RNA polymerase II as long transcripts which are then exported to the cytoplasm and processed into smaller sequences (mature piRNAs). In D. melanogaster, the nuclear export of piRNA precursors is mediated by UAP-56 that has been previously implicated in mRNA splicing and export. Another factor that has been important in the context of piRNAs biogenesis is the mitochondrial surface protein Zucchini. This protein has endonuclease activity for single-stranded RNA and likely processes the piRNA precursors, possibility at the 5′end. After 5′ processing, the 5′U/piRNA is incorporated into the MID domain of the Piwi protein, followed by 3′end processing, which likely involves an unknown trimmer activity as well as the action of methyl-transferase Pimet. Mature piRNAs are 5′ monophosphated and 2′-O-methyl modified in the 3′ terminal, a unique and specific feature of all piRNAs.
与 miRNA 和 siRNA 不同，piRNA 通常更长 (26-31 个核苷酸)，来源于单链 RNA 前体，并且成熟过程中不需要 Dicer (图 3)。大多数 piRNA 由 RNA 聚合酶 II 转录成长的转录本，然后运输到细胞质并加工成较小的序列 (成熟 piRNA)。在 D. melanogaster (黑腹果蝇) 中，piRNA 前体的运出核过程由 UAP-56 蛋白介导，UAP-56 蛋白之前被证实参与 mRNA 剪接和输出。线粒体表面蛋白 Zucchini 是另一个影响 piRNA 生物合成的重要因素。该蛋白具有单链 RNA 的核酸内切酶活性，可能加工 piRNA 前体 (特别是 5' 端)。经过 5' 端加工后，5'U/piRNA 被整合到 Piwi 蛋白的 MID 结构域中，然后进行 3' 端加工，这可能涉及未知的切割酶活性和甲基转移酶 Pimet 的作用。成熟的 piRNA 具有 5' 单磷酸酯基和 3' 末端 2'-O-甲基化修饰，这是所有 piRNA 的独特特征。
Fig. 3 Mechanisms of piRNA biogenesis in D. melanogaster. The piwi-interacting RNA pathway is involved in defense against transposable elements in the germline. Antisense precursor piRNAs are synthesized from repetitive elements by RNA polymerase II and then exported from the nucleus to nuage granules, where piRNA biogenesis is believed to occur. These piRNA precursors are cleaved by the endonuclease Zucchini, located in the outer membrane of mitochondria, generating the 5′end of the future piRNAs. Then they are trimmed to short fragments, and their 3′ ends are 2′-methylated and then loaded onto aubergine (Aub), which cleaves complementary transposon transcripts to generate sense piRNA fragments. Argonaute 3 (Ago3) uses the sense piRNA fragments to bind and cleave additional complementary antisense transposon transcripts to generate more antisense piRNAs, thus creating a feedback loop known as the ping-pong cycle. This amplification step generates new piRNAs that are identical in sequence to the piRNA that initiated the cycle.D. melanogaster中 piRNA 生物发生的机制。Piwi-相互作用的 RNA 通路参与生殖细胞系中对转座元件的防御。反义的 piRNA 前体是由 RNA 聚合酶 II 从重复序列转录合成,然后从核中被运输到 nuage 颗粒中,人们认为 piRNA 的生物合成过程发生在这里。这些 piRNA 前体被位于线粒体外膜上的内切酶 Zucchini 剪切,从而产生未来成熟 piRNA 的 5'端。接下来它们会被进一步修剪成更短的片段,并在 3'端发生 2'-甲基化修饰,然后装载到 aubergine(Aub)蛋白上。Aub 能够剪切与之互补的转座子转录本,从而产生正义的 piRNA 片段。Argonaute 3 (Ago3)则利用这些正义 piRNA 片段与其他互补的反义转座子转录本结合并剪切,产生更多的反义 piRNA,从而形成一个被称为"乒乓循环"的正反馈回路。这个扩增过程产生的新 piRNA 与启动该循环的初始 piRNA 序列是完全相同的。
The D. melanogaster genome encodes three Piwi proteins, Piwi, Aubergine (Aub), and Ago3, all of which are required for male and female fertility. As shown in Fig. 3, Piwi and Aub show a strong preference for sequences with a 5′uridine (U) within the antisense to transposons, whereas Ago3 shows enrichment for adenine (A) at position 10 within the sense to transposons, suggesting that Ago3 piRNAs are generated by a new mechanism known as the “ping-pong cycle” of amplification. In this cycle, Aub-bound piRNAs recognize a complementary transposon and induce endonuclease cleavage of the target. Such slicing generates the 5′ end of new sense piRNAs. This cytoplasmic loop, which is also found in zebrafish, links piRNA amplification to posttranscriptional target silencing. Additionally, mature piRNAs in the cytoplasm form a complex with Piwi proteins and migrate back into the nucleus, reaching their target transcripts and mobilizing the silencing machinery to block the transcription of transposable elements via DNA methylation (Fig. 3). Although the role of piRNAs in silencing transposon repeat elements is well established, recent reports have shown that piRNAs can also regulate gene expression and perfect sequence homology between the piRNA and its target mRNA is not required for efficient repression by deadenylation. Apart from the gonads, piRNAs and PIWI proteins may be expressed in various somatic cell types, albeit typically at lower levels than those observed in the germline . Similar to miRNAs , a higher number of piRNAs are expressed in tumors compared to normal tissues.
果蝇 (D. melanogaster) 基因组编码三种 Piwi 蛋白，分别是 Piwi、Aubergine (Aub) 和 Ago3，这三种蛋白质都对雄性和雌性繁殖力至关重要。如图 3 所示，Piwi 和 Aub 蛋白对转座子反义链中 5' 末端的尿苷 (U) 序列有强烈的偏好，而 Ago3 蛋白则对转座子顺义链中 10 位的腺苷 (A) 序列富集，这表明 Ago3 piRNA 是通过一种称为“乒乓循环”的扩增新机制产生的。在这个循环中，Aub 结合的 piRNA 识别互补的转座子并诱导靶标的核酸内切酶切割。这种切割产生新的顺义 piRNA 的 5' 端。这种循环也存在于斑马鱼身上，它将 piRNA 扩增与转录后靶标沉默联系起来。此外，细胞质中的成熟 piRNA 与 Piwi 蛋白结合并返回细胞核，到达靶向转录本并调动沉默机制，通过 DNA 甲基化阻止转座子元素的转录 (图 3)。尽管 piRNA 在沉默转座子重复序列方面的作用已得到充分证实，但最近的报道表明 piRNA 还可调控基因表达，并且 piRNA 与其靶向 mRNA 之间不需要完美的序列同源性即可通过使 mRNA 失去末端腺苷酸 (deadenylation) 而有效地抑制其表达。除了性腺之外，piRNA 和 PIWI 蛋白也可能在各种体细胞类型中表达，尽管表达水平通常低于生殖细胞中的水平。类似于 miRNA，肿瘤组织中表达的 piRNA 数量也高于正常组织。
