学院生命科学学院生物科学专业

1、学院：生命科学学院生物科学专业班级：0701姓名：段青学号：2007114010105REVIEW综述Many roads to maturity: 许多到期之路：microRNA biogenesis pathways and their regulation小分子RNA生物合成途径及其调控Julia Winter1,3,Stephanie Jung1,3, Sarina Keller1, Richard I. Gregory2 and Sven Diederichs1,4MicroRNAs are important regulators of gene expression that c

2、ontrol both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding

3、of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficie

4、ncy. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.小分子RNA是作为控制生长和癌症等生理和病理过程中基因表达的重要调节物。虽然他们的行为模式已引起高度重视，但控制它们表达和活动的原理才刚刚开始出现。最近的研究提出了在我们的理解范围内的微RNA合成途径的转变模式，这是过去所认同的

5、所有小分子RNA。针对个别小分子RNA的成熟步骤已被破获，这些为转录多种蛋白质后提供了很多影响小分子RNA处理效率的监管办法。在这里，我们回顾小分子RNA生物合成途径知识的最新进展，并讨论其在肿瘤发展过程中对转录后小分子RNA调控的影响。MicroRNAs (miRNAs) are short (2023-nucleotide), endogenous, single-stranded RNA molecules that regulate gene expression1. Mature miRNAs and Argonaute (Ago) proteins form the RNA-ind

6、uced silencing complex (RISC), a ribonucleoprotein complex mediating post-transcriptional gene silencing25. Complementary base-pairing of the miRNA guides RISC to target messenger RNAs, which are degraded, destabilized or translationally inhibited by the Ago protein6,7. 小分子RNA（miRNA的）短（20 - 23 -核苷酸）

7、，内源性，单链RNA分子，expression1的调节基因。成熟miRNAs与Argonaute (Ago) proteins形成RNA诱导沉默复合物(RISC)，是一种介导沉默基因转录的核蛋白复合体2-5。碱基互补性的miRNA引导RISC面向信使RNA，这是由Ago 蛋白引起的退化，不稳定或翻译后抑制6，7。 Proteomic studies have recently uncovered the broad impact of a single miRNA on hundreds of targets8,9. Many cellular pathways are affected by

8、 the regulatory function of miRNAs; the most prominent of these pathways control developmental and oncogenic processes1020. Notably, miRNA processing defects also enhance tumorigenesis21. Although insights intothe regulatory function of miRNAs are beginning to emerge, much less is known about the re

9、gulation of miRNA expression and activity. Recently, evidence for post-transcriptional control of miRNA activity has been accumulating2226. 蛋白质组研究最近发现了单一miRNA对于数百种靶标的广泛的影响8，9。许多细胞途径都受到了miRNA调节功能的影响，其中最突出的三条途径控制发展和致癌过程10 - 20。值得注意的是，miRNA的加工缺陷也提高肿瘤发生21。虽然对于miRNAs调节功能的见解已开始出现，但对miRNA的表达及活性调节却知之甚少。最近，

10、miRNA的转录后控制活动的证据正在增加。In contrast to the linear miRNA processing pathway that was initially thought to be universal for the biogenesis of all mature miRNAs (Fig. 1), multiple discoveries led to the recognition of miRNA-specific differences that open a plethora of regulatory options to express and pro

11、cess individual miRNAs differentially. Here we review the recent progress made in elucidating the complexity of miRNA processing and post-transcriptional regulation. Although we focus predominantly on the mammalian system, related information obtained from other model systems including the fruitfly

12、Drosophila melanogaster, the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana will also be presented where applicable.相对于线性miRNA的加工途径，最初认为是对所有成熟的miRNA的生物合成普遍的（图1），多发现其导致了对于miRNA特异性差异的认可，这打开了一套多样的表达和处理个体miRNA的差异的监管方案。在这里，我们回顾最近在阐明miRNA的处理的复合体性和转录后调控规律方面取得的进步。虽然我们把重点主要放在哺乳动物系统，但从包括果蝇

13、线虫和拟南芥植物等模式系统获得的相关信息也将在适当地方得到呈现。Early steps: microRNA processing in the nucleusTranscription of the pri-miRNA. miRNA genes are transcribed by either RNA polymerase II or RNA polymerase III into primary miRNA transcripts (pri-miRNA)2729. Many pri-miRNAs are polyadenylated and capped hallmarks of poly

14、merase II transcription. Their transcription is sensitive to treatment with the polymerase II inhibitor -amanitin, and polymerase II binds to promoter sequences upstream of the miR-23a/miR-27a/miR-24-2 cluster27,28. In contrast, miRNAs encoded by the largest human miRNA cluster, C19MC, are transcrib

15、ed by polymerase III29早期步骤：微RNA在细胞核中加工，翻译pri-miRNA, miRNA基因在RNA聚合酶II或RNA聚合酶III的催化作用下，被转录成初级miRNA副本（pri- miRNA的）27-29。许多pri- miRNA是呈多聚腺苷酸态的，并且是聚合酶II 催化转录的上限标志。它们的转录对于治疗聚合酶II抑制剂-鹅膏蕈碱是敏感的，并且聚合酶II结合上游miR-23a/miR-27a/miR-24-2 cluster27，28启动子序列。相比之下，人类最大的miRNA的集群，C19MC编码的miRNA，是由聚合酶III29转录的。Both RNA polym

16、erases are regulated differently and recognize specific promoter and terminator elements, facilitating a wide variety of regulatory options. Expression of selected miRNAs is under the control of transcription factors, for example c-Myc or p53 (refs 17, 19), or depends on the methylation of their pro

17、moter sequences3032. In addition, it has been shown that each miRNA located in the same genomic cluster can be transcribed and regulated independently33. However, controls of miRNA transcription steps are not necessarily universal 34,35, and regulatory mech-anisms at the transcriptional level are be

18、yond the scope of this review.两种RNA聚合酶受不同机制的调节，并识别特定启动子和终止元素，促进了多种调控选择。选定的miRNA的表达受转录因子控制，例如c - Myc基因或p53（参17，19），或依赖于其子序列30- 32的甲基化作用。此外，它已表明，位于相同基因簇的每个miRNA能被33独立的转录和调控。然而，miRNA转录步骤的控制不是普遍必需的34,35，转录水平的监管机制已超出了本综述的范围。microRNA editing. RNA editing of primary transcripts by ADARs (adenosine deaminas

19、es acting on RNA) modifies adenosine (A) into inosine (I). Because the base-pairing properties of inosine are similar to those of guanosine (G), A-to-I editing of miRNA precursors may change their sequence, base-pairing and structural properties and can influence their further processing as well as

20、their target recognition abilities. Several examples of editing-mediated regulation of miRNA processing have been described (see Box 1).RNA编辑的初级转录本由ADARs（RNA的作用腺苷deaminases）将腺苷（A)修改成肌苷（I）。由于肌苷的碱基配对与鸟苷（G）的相似，A到I miRNA前体的编辑可能会改变它们的序列，碱基配对及结构特性，并影响他们更深层次的加工以及他们的目标识别能力。有几个编辑介导miRNA加工调控的例子曾有人描述过了（见专栏1）。1

21、Helmholtz-University-Group Molecular RNA Biology & Cancer, German Cancer Research Center (DKFZ) and Institute of Pathology, University of Heidelberg, B150 INF 581, D-69120 Heidelberg, Germany. 2Stem Cell Program, Childrens Hospital Boston, Department of Biological Chemistry and Molecular Pharmacolog

22、y, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts 02115, USA. 3These authors contributed equally to the work. 4Correspondence should be addressed to S.D. (e-mail: s.diederichsdkfz.de)1亥姆霍兹大学组分子RNA生物学与癌症，德国癌症研究中心（DKFZ）和病理学研究所，海德堡大学，B150 INF 581，D-69120海德尔堡，德国。 2干细胞研究项目，波士顿

23、儿童医院，生物化学与分子药理学，哈佛医学院，哈佛干细胞研究所，波士顿，马萨诸塞州02115，美国。 3这些作者对这项工作有同等的贡献。4来信请寄S.D.（电子邮箱:s.diederichsdkfz.de）Figure 1 The linear canonical pathway of microRNA processing. The miRNA processing pathway has long been viewed as linear and universal to all mammalian miRNAs. This canonical includes the productio

24、n of the primary miRNA transcript (pri-miRNA) by RNA polymerase II or III and cleavage of the pri-miRNA by the microprocessor complex DroshaDGCR8 (Pasha) in the nucleus. The resulting precursor hairpin, the pre-miRNA, is exported from the nucleus by Exportin-5Ran-GTP. In the cytoplasm, the RNase Dic

25、er in complex with the double-stranded RNA-binding protein TRBP cleaves the pre-miRNA hairpin to its mature length. The functional strand of the mature miRNA is loaded together with Argonaute (Ago2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target mRNAs

26、through mRNA cleavage, translational repression or deadenylation, whereas the passenger strand (black) is degraded. In this review we discuss the many branches, crossroads and detours in miRNA processing that lead to the conclusion that many different ways exist to generate a mature miRNA.图1小分子RNA加工

27、的线性规范途径。miRNA的加工途径长期以来被视为线性的并普及到所有哺乳动物miRNA。本规范包括由RNA聚合酶II或III介导的初级miRNA（PRI - miRNA）副本产物和由细胞核中的微处理器复合物DroshaDGCR8 (Pasha)介导的pri-miRNA的裂解。由此产生的前体发夹，即前miRNA，是由Exportin-5Ran-GTP介导输出细胞核的。在细胞质中，由双链RNA结合蛋白TRBP构成的复合体物核糖核酸酶Dicer诱导前miRNA发夹裂开到其成熟长度。成熟miRNA的功能链与Argonaute（Ago2）蛋白一起被加载到RNA诱导沉默复合体（RISC）上，在那里通过mR

28、NA的切割，翻译抑制或脱腺苷化作用后引导RISC到沉默靶mRNA，然而passenger链（黑）是退化的。在这篇综述中，我们讨论了许多分支，在得出miRNA加工结论方面走了很多弯路，这一结论是：产生一个成熟的miRNA存在许多不同的方式。pri-miRNA cleavage by the DroshaDGCR8 microprocess or complex. The pri-miRNA is next endonucleoly tically cleaved by the nuclear micro-processor complex formed by the RNase III enzy

29、me Drosha (RNASEN) and the DGCR8 (DiGeorge critical region 8) protein (also known as Pasha (Partner of Drosha) in D. melanogaster and C. elegans)36 (Fig. 2a). DGCR8/Pasha contains two double-stranded RNA-binding domains and is essential for miRNA processing in all organisms tested 3740.由Drosha - DGC

30、R8微处理器复合体介导的pri-miRNA的分裂。前miRNA 由附近的核糖核酸酶IIIDrosha（RNASEN）和DGCR8（DiGeorge critical region 8）蛋白（在D. 果蝇和C.线虫中也称为Pasha(Partner of Drosha)）组成核微处理器复合体进行旋光切割（图2a）36。 DGCR8/Pasha含有两个双链RNA结合域，并且对于所有被测有机体的miRNA加工是必不可少的37-40。 An average human pri-miRNA contains a hairpin stem of 33 base-pairs, a terminal loop

31、 and two single-stranded flanking regions upstream and downstream of the hairpin. The double-stranded stem and the unpaired flanking regions are critical for DGCR8 binding and Drosha cleavage, but the loop region or the specific sequences are less important for this step 4143. A single nucleotide po

32、lymorphism in a miRNA precursor stem can block Drosha processing 44. Nevertheless, many miRNA sequence aberrations observed in human tumours alter the secondary structure without affecting processing, and reveal the structural flexibility of the microprocessor 34. 正常人pri-miRNA包含一个由33个碱基对组成的发夹干，一个终端环

33、路和两个位于发夹干上游和下游的单链侧翼区。双链茎和未成对侧翼区对于DGCR8的约束和Drosha的切割是至关重要的，但循环区和特定序列对于这一步是次要的41 43。位于miRNA前体干细胞的单核苷酸多态性可以阻止Drosha加工44。然而，许多在人体肿瘤中发现的二级结构的改变引起的miRNA序列畸变不影响加工过程，从而揭示了微处理器的结构灵活性34。The two RNase domains of Drosha cleave the 5 and 3 arms of the pri-miRNA hairpin39, whereas DGCR8 directly and stably intera

34、cts with the pri-miRNA and functions as a molecular ruler to determine the precise cleavage site 41. Drosha cleaves 11 base pairs away from the single-stranded RNA/double-stranded RNA junction at the base of the hairpin stem. Drosha-mediated cleavage of the pri-miRNA occurs co-transcriptionally and

35、precedes splicing of the protein-encoding or non-coding host RNA hat contains the miRNAs. Splicing is not inhibited by Drosha-mediated cleavage, because a continuous intron is not required for splicing 45,46.Drosha的两个酶域切割pri-miRNA发夹的5端和3端39，而DGCR8直接、稳定地作用于pri-miRNA，并作为分子标尺准确确定裂解位点41。 Drosha从单链RNA /双

36、链RNA发夹干基交界处解离出11个碱基对。 Drosha介导的pri-miRNA裂解发生合作转录，并且先于蛋白编码的剪接，或非编码RNA帽子结构含有miRNA。拼接不被Drosha介导的切割所抑制，因为拼接不需连续的内含子45，46。microRNA-specific regulation of the microprocessor complex. Drosha-mediated pri-miRNA processing was recently shown to be subject to regulation by miRNA-specific mechanisms. Drosha fo

37、rms different complexes, a small microprocessor complex that contains only Drosha and DGCR8 and processes many pri-miRNAs, and a larger complex that contains RNA helicases, double-stranded RNA binding proteins, heterogeneous nuclear ribonucleoproteins and Ewings sarcoma proteins38. 小分子RNA微处理器复合体的特定调

38、控规则。 Drosha介导的pri- miRNA的加工最近证明须经miRNA特定机制的调节。 Drosha形式不同的复合物，一个小型微处理器复合体仅包含Drosha和DGCR8并加工许多pri-miRNA，而一个大型的复合体包含RNA解旋酶，双链RNA结合蛋白，异构核核糖和Ewings肉瘤蛋白38。The RNA helicases p72 and p68 are part of the large Drosha complex and might act as specificity factors for the processing of a subset of pri-miRNAs (

39、Fig. 2b). Expression levels of several miRNAs are reduced in homozygous p68/ or p72/ knockout mice, whereas other miRNAs remain unaffected 47. RNA解旋酶p72和p68是大型Drosha复合体的一部分，并有可能作为介导pri-miRNAs加工的特殊因子而起作用（图2b）。个别miRNAs在纯p68 - /- 或p72 - / - 基因敲除小鼠中的表达水平是被减弱的，然而其他miRNA不受影响47。 Drosha-mediated cleavage ca

40、n also be regulated for individual miRNAs: the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds specifically to pri-miR-18a and facilitates its processing. Loss of hnRNP A1 diminishes the abundance of mature miR-18a (Fig. 2c), but hnRNP A1 does not have any impact on other miRNAs that are

41、 located the same miR-17 genomic cluster, demonstrating the extraordinary pecificity of miR-18a biogenesis 48. hnRNP A1 binds to the conserved oop of the pri-miR-18a and changes the hairpin conformation to create a more favourable cleavage site for Drosha49. About 14% of the human pri-miRNA loops ar

42、e conserved between different species and could provide anchor points for similar regulatory mechanisms. Transforming growth factor- (TGF-) and bone morphogenetic actors (BMPs) induce the maturation of miR-21 by regulating the microprocessor activity. TGF- and BMP bring about the recruitment.Drosha介

43、导的切割也可以调节个别miRNA：异构核核糖核蛋白 A1（hnRNP A1A1）特异性结合pri-miR-18a，并促进其处理。hnRNP A1的丢失削弱了大量成熟miR-18a（图2C型），但hnRNP A1对于位于相同miR - 17基因组上的其它miRNA没有任何影响，这表明了miR-18a 生物合成的非凡特异性48。hnRNP A1结合pri-miR-18a的保守oop并将发夹构象变为一个更有利的Drosha 49裂解位点。约14的人体pri-miRNA循环结构在不同物种间是保守的，可提供类似规管机制的锚定点。转化生长因子-（TGF-）和骨形态建成因子(BMPs)通过调节微处理器活动诱

44、导miR-21的成熟。由TGF-和BMP对其进行补充。Editing can influence further downstream processing steps: pri-miR-151 editing abolishes its cleavage by Dicer in the cyto-plasm. It remains to be established whether miRNA editing events are predominantly nuclear or cytoplasmic and whether they occur on the pri-miRNA or o

45、n the precursor miRNA (pre-miRNA)121. In addition to altering miRNA processing, miRNA editing can have an impact on miRNA target specificity. For example, a single A-to-I change in the miR-376 precursor redirects the mature miRNA to a new target, resulting in altered protein expression in mice 122.

46、编辑可以进一步影响下游加工步骤：pri-miR-151编辑由巨细胞浆中的Dicer酶取消其切割。miRNA的编辑事件是否主要在核或细胞质中和它们是否发生在pri-miRNA或前体miRNA（pre-miRNA）121上仍有待确定。除了改变miRNA的加工，miRNA的编辑也可影响miRNA的靶标特异性。例如，在miR - 376前体中由单个A到I的改变使成熟miRNA重定向到一个新的目标，导致小鼠122蛋白表达的改变。In summary, miRNA editing can influence processing at multiple steps or can change the mi

47、RNA complementarity to target sequences, increasing the diversity of the cellular miRNA pool. BOX 1 microRNA editing of ligand-specific signal transducers (the SMAD proteins) to the pri-miR-21 transcript in complex with the RNA helicase DDX5 (p68). As a consequence, Drosha-mediated processing of pri

48、-miR-21 is strongly enhanced and the abundance of mature miR-21 increases, ultimately resulting in a contractile phenotype in vascular smooth muscle cells (Fig. 2d)50总之，miRNA的编辑可以在多个步骤影响加工处理，或可以改变miRNA靶序列的互补性，从而增加的细胞miRNA库的多样性。专栏1小分子RNA编辑的配体特异性信号（即SMAD蛋白）对pri-miR-21的副本与RNA解旋酶DDX5的复合体进行转导（p68）。因此，Dro

49、sha介导的pri-miR-21的处理被大大加强，大量成熟miR - 21增加，最终导致血管平滑肌细胞收缩表型（图二维）50。Mirtrons: splicing replaces Drosha cleavage. Surprisingly, Drosha-mediated processing of pri-miRNAs into pre-miRNAs is not obligatory. Intron-derived miRNAs are released from their host transcripts after splicing (Fig. 2e). If the intron

50、 resulting from the action of the splicing machinery and the lariat debranching enzyme has the appropriate size to form a hairpin resembling a pre-miRNA, it bypasses Drosha cleavage and is further processed in the cytoplasm by Dicer 51,52. These miRNAs, called mirtrons, have been discovered in sever

51、al species including mam-mals, D. melanogaster and C. elegans5153.Mirtrons：剪接取代Drosha切割。令人惊讶的是，Drosha介导的pri-miRNA向pre-miRNA的加工处理不是强制性的。内含子衍生miRNA在拼接之后从它们的主副本中被释放出来（图2e）。如果由剪接机制和lariat支酶产生的内含子具有适当的规模来形成似发夹结构的pre-miRNA，它绕过Drosha切割，并在细胞质中由Dicer51，52作进一步处理。这些被称为mirtrons 的miRNA，已在包括哺乳类物种mals，D. melanogas

52、ter和线虫51-53中被发现。Lin-28 regulates let-7 processing and precursor stability. Lin-28 is a stem-cell-specific regulator of let-7 processing that uses multiple mechanisms 5458. Lin-28 was found to be necessary and sufficient to block microprocessor-mediated cleavage of the pri-miRNA (Fig. 3a)54. Lin-28调节

53、let - 7的处理和前体的稳定。Lin-28是一种使用多发性机制的干细胞特异性let - 7处理调节器 54-58。Lin-28对于阻止微处理器介导的pri-miRNA切割是必要和充分的（图3a）54。Mature let-7g increases during embryonic stem cell differentiation but the pri-miRNA levels remain constant, indicating post-transcriptional regulation of maturation. Recombinant Lin-28 blocks pri-m

54、iRNA processing, and knockdown of Lin-28 facilitates the expression of mature let-7 (ref. 54). The miRNA binding site of the Drosha competitor Lin-28 maps to conserved bases in the ter-minal loop of pri-let-7 (refs 56, 57). Intriguingly, although the loop region is considered dispensable for micropr

55、ocessor action, many miRNAs have evolutionarily conserved loops potentially containing regulatory information49. 成熟let-7g在胚胎干细胞分化期间上升，但pri-miRNA水平保持不变，预示后转录成熟调节。重组Lin-28阻止pri-miRNA的加工，并阻止Lin-28催化成熟let - 7的表达（参见54）。miRNA上Drosha和Lin-28的竞争性结合位点保守地以三末端循环的pri-let-7为基础（参56，57）。耐人寻味的是，虽然环区被认为是可有可无的微处理器行为，但

56、许多miRNA拥有可能包含管理信息49的在进化上保守的循环区。 Post-transcriptional self-regulation of the microprocessor complex. The miRNA processing factors are also regulated post-transcription-ally or post-translationally. For example, the two components of the microprocessor complex regulate each other. DGCR8 stabilizes Dro

57、sha through an interaction between its conserved carboxy-terminal domain with the middle domain of Drosha (Fig. 4a)59. In turn, Drosha cleaves two hairpin structures in the 5 untranslated region and the coding sequence of the Dgcr8 mRNA60. The Dgcr8 mRNA is then degraded, resulting in a negative fee

58、dback loop reducing Dgcr8 expression when sufficient microprocessor activity is available (Fig. 4b). The discovery that Drosha can directly cleave hairpin structures in mRNAs also points to the possibility that the two Drosha complexes in the cell regulate mRNAs independently of miRNAs. 转录后微处理器复合体的自

59、我调节。 miRNA的加工因素也在转录后或翻译后被调节。例如，微处理器复合体的两个组成部分互相调节。 DGCR8通过保守羧基末端结构域与Drosha中域相互作用稳定Drosha（图4A）59。反过来，Drosha劈开5非编码区两个发夹结构和Dgcr8 mRNA60编码序列。Dgcr8 mRNA然后退化，造成当微处理器活动时负反馈循环减少Dgcr8的表达（图4b）。Drosha可以直接切割mRNA中的发夹结构这一发现也指出了细胞中的两个Drosha复合物独立调节miRNA 的mRNA的可能性。Exportin-5Ran-GTP mediate the export of the pre-miRNA. After nuclear processing, the pre-miRNA