1、学院生命科学学院生物科学专业6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 ex
2、pression that control 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
3、 understanding 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 pro
4、cessing efficiency. 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
5、径的转变模式，这是过去所认同的所有小分子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 f
6、orm the RNA-induced 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）
7、短（20 - 23 -核苷酸），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
8、are affected by 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 kno
9、wn about the regulation 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
10、及活性调节却知之甚少。最近， 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
11、express and process 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 includin
12、g the fruitfly 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
13、放在哺乳动物系统，但从包括果蝇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 ha
14、llmarks of polymerase 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
15、, are transcribed 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
16、。Both RNA polymerases 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 methylati
17、on of their promoter 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 transcription
18、al level are beyond 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 (ade
19、nosine deaminases 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 process
20、ing as well as 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
21、曾有人描述过了（见专栏1）。1Helmholtz-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 Molecu
22、lar Pharmacology, 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
23、国。 2干细胞研究项目，波士顿儿童医院，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 include
24、s the production 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
25、, the RNase Dicer 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 silenc
26、e target mRNAs 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 m
27、iRNA.图1小分子RNA加工的线性规范途径。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
28、（RISC）上，在那里通过mRNA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
29、 RNase III enzyme 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 374
30、0.由Drosha - DGCR8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,
31、a terminal loop 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 singl
32、e nucleotide polymorphism 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
