學(xué)院生命科學(xué)學(xué)院生物科學(xué)專業(yè)

1、學(xué)院：生命科學(xué)學(xué)院生物科學(xué)專業(yè)班級(jí)：0701姓名：段青學(xué)號(hào)：2007114010105REVIEW綜述Many roads to maturity: 許多到期之路：microRNA biogenesis pathways and their regulation小分子RNA生物合成途徑及其調(diào)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是作為控制生長(zhǎng)和癌癥等生理和病理過(guò)程中基因表達(dá)的重要調(diào)節(jié)物。雖然他們的行為模式已引起高度重視，但控制它們表達(dá)和活動(dòng)的原理才剛剛開(kāi)始出現(xiàn)。最近的研究提出了在我們的理解范圍內(nèi)的微RNA合成途徑的轉(zhuǎn)變模式，這是過(guò)去所認(rèn)同的

5、所有小分子RNA。針對(duì)個(gè)別小分子RNA的成熟步驟已被破獲，這些為轉(zhuǎn)錄多種蛋白質(zhì)后提供了很多影響小分子RNA處理效率的監(jiān)管辦法。在這里，我們回顧小分子RNA生物合成途徑知識(shí)的最新進(jìn)展，并討論其在腫瘤發(fā)展過(guò)程中對(duì)轉(zhuǎn)錄后小分子RNA調(diào)控的影響。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、，內(nèi)源性，單鏈RNA分子，expression1的調(diào)節(jié)基因。成熟miRNAs與Argonaute (Ago) proteins形成RNA誘導(dǎo)沉默復(fù)合物(RISC)，是一種介導(dǎo)沉默基因轉(zhuǎn)錄的核蛋白復(fù)合體2-5。堿基互補(bǔ)性的miRNA引導(dǎo)RISC面向信使RNA，這是由Ago 蛋白引起的退化，不穩(wěn)定或翻譯后抑制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. 蛋白質(zhì)組研究最近發(fā)現(xiàn)了單一miRNA對(duì)于數(shù)百種靶標(biāo)的廣泛的影響8，9。許多細(xì)胞途徑都受到了miRNA調(diào)節(jié)功能的影響，其中最突出的三條途徑控制發(fā)展和致癌過(guò)程10 - 20。值得注意的是，miRNA的加工缺陷也提高腫瘤發(fā)生21。雖然對(duì)于miRNAs調(diào)節(jié)功能的見(jiàn)解已開(kāi)始出現(xiàn)，但對(duì)miRNA的表達(dá)及活性調(diào)節(jié)卻知之甚少。最近，

10、miRNA的轉(zhuǎn)錄后控制活動(dòng)的證據(jù)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.相對(duì)于線性miRNA的加工途徑，最初認(rèn)為是對(duì)所有成熟的miRNA的生物合成普遍的（圖1），多發(fā)現(xiàn)其導(dǎo)致了對(duì)于miRNA特異性差異的認(rèn)可，這打開(kāi)了一套多樣的表達(dá)和處理個(gè)體miRNA的差異的監(jiān)管方案。在這里，我們回顧最近在闡明miRNA的處理的復(fù)合體性和轉(zhuǎn)錄后調(diào)控規(guī)律方面取得的進(jìn)步。雖然我們把重點(diǎn)主要放在哺乳動(dòng)物系統(tǒng)，但從包括果蠅

13、線蟲和擬南芥植物等模式系統(tǒng)獲得的相關(guān)信息也將在適當(dāng)?shù)胤降玫匠尸F(xiàn)。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早期步驟：微RNA在細(xì)胞核中加工，翻譯pri-miRNA, miRNA基因在RNA聚合酶II或RNA聚合酶III的催化作用下，被轉(zhuǎn)錄成初級(jí)miRNA副本（pri- miRNA的）27-29。許多pri- miRNA是呈多聚腺苷酸態(tài)的，并且是聚合酶II 催化轉(zhuǎn)錄的上限標(biāo)志。它們的轉(zhuǎn)錄對(duì)于治療聚合酶II抑制劑-鵝膏蕈堿是敏感的，并且聚合酶II結(jié)合上游miR-23a/miR-27a/miR-24-2 cluster27，28啟動(dòng)子序列。相比之下，人類最大的miRNA的集群，C19MC編碼的miRNA，是由聚合酶III29轉(zhuǎn)錄的。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.兩種RNA聚合酶受不同機(jī)制的調(diào)節(jié)，并識(shí)別特定啟動(dòng)子和終止元素，促進(jìn)了多種調(diào)控選擇。選定的miRNA的表達(dá)受轉(zhuǎn)錄因子控制，例如c - Myc基因或p53（參17，19），或依賴于其子序列30- 32的甲基化作用。此外，它已表明，位于相同基因簇的每個(gè)miRNA能被33獨(dú)立的轉(zhuǎn)錄和調(diào)控。然而，miRNA轉(zhuǎn)錄步驟的控制不是普遍必需的34,35，轉(zhuǎn)錄水平的監(jiān)管機(jī)制已超出了本綜述的范圍。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編輯的初級(jí)轉(zhuǎn)錄本由ADARs（RNA的作用腺苷deaminases）將腺苷（A)修改成肌苷（I）。由于肌苷的堿基配對(duì)與鳥苷（G）的相似，A到I miRNA前體的編輯可能會(huì)改變它們的序列，堿基配對(duì)及結(jié)構(gòu)特性，并影響他們更深層次的加工以及他們的目標(biāo)識(shí)別能力。有幾個(gè)編輯介導(dǎo)miRNA加工調(diào)控的例子曾有人描述過(guò)了（見(jiàn)專欄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 Pharmac

22、ology, 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亥姆霍茲大學(xué)組'分子RNA生物學(xué)與癌癥'，德國(guó)癌癥研究中心（DKFZ）和病理學(xué)研究所，海德堡大學(xué)，B150 INF 581，D-69120海德?tīng)柋?，德?guó)

23、。 2干細(xì)胞研究項(xiàng)目，波士頓兒童醫(yī)院，生物化學(xué)與分子藥理學(xué)，哈佛醫(yī)學(xué)院，哈佛干細(xì)胞研究所，波士頓，馬薩諸塞州02115，美國(guó)。 3這些作者對(duì)這項(xiàng)工作有同等的貢獻(xiàn)。4來(lái)信請(qǐng)寄S.D.（電子郵箱: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

24、 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 silence

26、 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 mi

27、RNA.圖1小分子RNA加工的'線性'規(guī)范途徑。miRNA的加工途徑長(zhǎng)期以來(lái)被視為線性的并普及到所有哺乳動(dòng)物miRNA。本規(guī)范包括由RNA聚合酶II或III介導(dǎo)的初級(jí)miRNA（PRI - miRNA）副本產(chǎn)物和由細(xì)胞核中的微處理器復(fù)合物DroshaDGCR8 (Pasha)介導(dǎo)的pri-miRNA的裂解。由此產(chǎn)生的前體發(fā)夾，即前miRNA，是由Exportin-5Ran-GTP介導(dǎo)輸出細(xì)胞核的。在細(xì)胞質(zhì)中，由雙鏈RNA結(jié)合蛋白TRBP構(gòu)成的復(fù)合體物核糖核酸酶Dicer誘導(dǎo)前miRNA發(fā)夾裂開(kāi)到其成熟長(zhǎng)度。成熟miRNA的功能鏈與Argonaute（Ago2）蛋白一起被加載到R

28、NA誘導(dǎo)沉默復(fù)合體（RISC）上，在那里通過(guò)mRNA的切割，翻譯抑制或脫腺苷化作用后引導(dǎo)RISC到沉默靶mRNA，然而passenger鏈（黑）是退化的。在這篇綜述中，我們討論了許多分支，在得出miRNA加工結(jié)論方面走了很多彎路，這一結(jié)論是：產(chǎn)生一個(gè)成熟的miRNA存在許多不同的方式。pri-miRNA cleavage by the DroshaDGCR8 microprocess or complex. The pri-miRNA is next endonucleoly tically cleaved by the nuclear micro-processor complex form

29、ed by the 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 t

30、ested 3740.由Drosha - DGCR8微處理器復(fù)合體介導(dǎo)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. 果蠅和C.線蟲中也稱為Pasha(Partner of Drosha)）組成核微處理器復(fù)合體進(jìn)行旋光切割（圖2a）36。 DGCR8/Pasha含有兩個(gè)雙鏈RNA結(jié)合域，并且對(duì)于所有被測(cè)有機(jī)體的miRNA加工是必不可少的37-40。 An average human pri-miRNA contains a hairpin stem of 33 bas

31、e-pairs, 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

32、. A single 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-miRN

33、A包含一個(gè)由33個(gè)堿基對(duì)組成的發(fā)夾干，一個(gè)終端環(huán)路和兩個(gè)位于發(fā)夾干上游和下游的單鏈側(cè)翼區(qū)。雙鏈莖和未成對(duì)側(cè)翼區(qū)對(duì)于DGCR8的約束和Drosha的切割是至關(guān)重要的，但循環(huán)區(qū)和特定序列對(duì)于這一步是次要的41 43。位于miRNA前體干細(xì)胞的單核苷酸多態(tài)性可以阻止Drosha加工44。然而，許多在人體腫瘤中發(fā)現(xiàn)的二級(jí)結(jié)構(gòu)的改變引起的miRNA序列畸變不影響加工過(guò)程，從而揭示了微處理器的結(jié)構(gòu)靈活性34。The two RNase domains of Drosha cleave the 5´ and 3´ arms of the pri-miRNA hairpin39, wher

34、eas DGCR8 directly and stably interacts 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-mi

35、RNA occurs co-transcriptionally and 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的兩個(gè)酶域切割pri-miRNA發(fā)夾的5'端和3'端39，而DGCR8直接、穩(wěn)定地作

36、用于pri-miRNA，并作為分子標(biāo)尺準(zhǔn)確確定裂解位點(diǎn)41。 Drosha從單鏈RNA /雙鏈RNA發(fā)夾干基交界處解離出11個(gè)堿基對(duì)。 Drosha介導(dǎo)的pri-miRNA裂解發(fā)生合作轉(zhuǎn)錄，并且先于蛋白編碼的剪接，或非編碼RNA帽子結(jié)構(gòu)含有miRNA。拼接不被Drosha介導(dǎo)的切割所抑制，因?yàn)槠唇硬恍柽B續(xù)的內(nèi)含子45，46。microRNA-specific regulation of the microprocessor complex. Drosha-mediated pri-miRNA processing was recently shown to be subject to regu

37、lation by miRNA-specific mechanisms. Drosha forms 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 an

38、d Ewings sarcoma proteins38. 小分子RNA微處理器復(fù)合體的特定調(diào)控規(guī)則。 Drosha介導(dǎo)的pri- miRNA的加工最近證明須經(jīng)miRNA特定機(jī)制的調(diào)節(jié)。 Drosha形式不同的復(fù)合物，一個(gè)小型微處理器復(fù)合體僅包含Drosha和DGCR8并加工許多pri-miRNA，而一個(gè)大型的復(fù)合體包含RNA解旋酶，雙鏈RNA結(jié)合蛋白，異構(gòu)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

39、for the processing of a subset of pri-miRNAs (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復(fù)合體的一部分，并有可能作為介導(dǎo)pri-miRNAs加工的特殊因子而起作用（圖2b）。個(gè)別miRNAs在純p68 - /- 或p72 - / - 基因敲除小鼠中的表達(dá)水平是被減弱

40、的，然而其他miRNA不受影響47。 Drosha-mediated cleavage can 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 doe

41、s not have any impact on other miRNAs that are 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 Drosh

42、a49. About 14% of the human pri-miRNA loops are 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. TG

43、F- and BMP bring about the recruitment.Drosha介導(dǎo)的切割也可以調(diào)節(jié)個(gè)別miRNA：異構(gòu)核核糖核蛋白 A1（hnRNP A1A1）特異性結(jié)合pri-miR-18a，并促進(jìn)其處理。hnRNP A1的丟失削弱了大量成熟miR-18a（圖2C型），但hnRNP A1對(duì)于位于相同miR - 17基因組上的其它miRNA沒(méi)有任何影響，這表明了miR-18a 生物合成的非凡特異性48。hnRNP A1結(jié)合pri-miR-18a的保守oop并將發(fā)夾構(gòu)象變?yōu)橐粋€(gè)更有利的Drosha 49裂解位點(diǎn)。約14的人體pri-miRNA循環(huán)結(jié)構(gòu)在不同物種間是保守的，可提供類似規(guī)

44、管機(jī)制的錨定點(diǎn)。轉(zhuǎn)化生長(zhǎng)因子-（TGF-）和骨形態(tài)建成因子(BMPs)通過(guò)調(diào)節(jié)微處理器活動(dòng)誘導(dǎo)miR-21的成熟。由TGF-和BMP對(duì)其進(jìn)行補(bǔ)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 cytoplasmi

45、c and whether they occur on the pri-miRNA or on 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, resulti

46、ng in altered protein expression in mice 122. 編輯可以進(jìn)一步影響下游加工步驟：pri-miR-151編輯由巨細(xì)胞漿中的Dicer酶取消其切割。miRNA的編輯事件是否主要在核或細(xì)胞質(zhì)中和它們是否發(fā)生在pri-miRNA或前體miRNA（pre-miRNA）121上仍有待確定。除了改變miRNA的加工，miRNA的編輯也可影響miRNA的靶標(biāo)特異性。例如，在miR - 376前體中由單個(gè)A到I的改變使成熟miRNA重定向到一個(gè)新的目標(biāo)，導(dǎo)致小鼠122蛋白表達(dá)的改變。In summary, miRNA editing can influence pro

47、cessing at multiple steps or can change the miRNA 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

48、consequence, Drosha-mediated processing of pri-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總之，miRNA的編輯可以在多個(gè)步驟影響加工處理，或可以改變miRNA靶序列的互補(bǔ)性，從而增加的細(xì)胞miRNA庫(kù)的多樣性。專欄1小分子RNA編輯的配體特異性信號(hào)（即SMAD蛋白

49、）對(duì)pri-miR-21的副本與RNA解旋酶DDX5的復(fù)合體進(jìn)行轉(zhuǎn)導(dǎo)（p68）。因此，Drosha介導(dǎo)的pri-miR-21的處理被大大加強(qiáng)，大量成熟miR - 21增加，最終導(dǎo)致血管平滑肌細(xì)胞收縮表型（圖二維）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 trans

50、cripts after splicing (Fig. 2e). If the intron 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,

51、called mirtrons, have been discovered in several species including mam-mals, D. melanogaster and C. elegans5153.Mirtrons：剪接取代Drosha切割。令人驚訝的是，Drosha介導(dǎo)的pri-miRNA向pre-miRNA的加工處理不是強(qiáng)制性的。內(nèi)含子衍生miRNA在拼接之后從它們的主副本中被釋放出來(lái)（圖2e）。如果由剪接機(jī)制和lariat支酶產(chǎn)生的內(nèi)含子具有適當(dāng)?shù)囊?guī)模來(lái)形成似發(fā)夾結(jié)構(gòu)的pre-miRNA，它繞過(guò)Drosha切割，并在細(xì)胞質(zhì)中由Dicer51，52作進(jìn)一步處理。這

52、些被稱為mirtrons 的miRNA，已在包括哺乳類物種mals，D. melanogaster和線蟲51-53中被發(fā)現(xiàn)。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 c

53、leavage of the pri-miRNA (Fig. 3a)54. Lin-28調(diào)節(jié)let - 7的處理和前體的穩(wěn)定。Lin-28是一種使用多發(fā)性機(jī)制的干細(xì)胞特異性let - 7處理調(diào)節(jié)器 54-58。Lin-28對(duì)于阻止微處理器介導(dǎo)的pri-miRNA切割是必要和充分的（圖3a）54。Mature let-7g increases during embryonic stem cell differentiation but the pri-miRNA levels remain constant, indicating post-transcriptional regulation

54、of maturation. Recombinant Lin-28 blocks pri-miRNA 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 loo

55、p region is considered dispensable for microprocessor action, many miRNAs have evolutionarily conserved loops potentially containing regulatory information49. 成熟let-7g在胚胎干細(xì)胞分化期間上升，但pri-miRNA水平保持不變，預(yù)示后轉(zhuǎn)錄成熟調(diào)節(jié)。重組Lin-28阻止pri-miRNA的加工，并阻止Lin-28催化成熟let - 7的表達(dá)（參見(jiàn)54）。miRNA上Drosha和Lin-28的競(jìng)爭(zhēng)性結(jié)合位點(diǎn)保守地以三末端循環(huán)的pri

56、-let-7為基礎(chǔ)（參56，57）。耐人尋味的是，雖然環(huán)區(qū)被認(rèn)為是可有可無(wú)的微處理器行為，但許多miRNA擁有可能包含管理信息49的在進(jìn)化上保守的循環(huán)區(qū)。 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 com

57、plex regulate each other. DGCR8 stabilizes Drosha 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 Dgcr

58、8 mRNA is then degraded, resulting in a negative feedback 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

59、regulate mRNAs independently of miRNAs. 轉(zhuǎn)錄后微處理器復(fù)合體的自我調(diào)節(jié)。 miRNA的加工因素也在轉(zhuǎn)錄后或翻譯后被調(diào)節(jié)。例如，微處理器復(fù)合體的兩個(gè)組成部分互相調(diào)節(jié)。 DGCR8通過(guò)保守羧基末端結(jié)構(gòu)域與Drosha中域相互作用穩(wěn)定Drosha（圖4A）59。反過(guò)來(lái)，Drosha劈開(kāi)5'非編碼區(qū)兩個(gè)發(fā)夾結(jié)構(gòu)和Dgcr8 mRNA60編碼序列。Dgcr8 mRNA然后退化，造成當(dāng)微處理器活動(dòng)時(shí)負(fù)反饋循環(huán)減少Dgcr8的表達(dá)（圖4b）。Drosha可以直接切割mRNA中的發(fā)夾結(jié)構(gòu)這一發(fā)現(xiàn)也指出了細(xì)胞中的兩個(gè)Drosha復(fù)合物獨(dú)立調(diào)節(jié)miRNA 的mRNA的可能性。Exportin-5Ran-GTP mediate the export of the pre-miRNA. After nuclear processing, the pre-miRNA