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1、非编码RNA与RNA组学NoncodingRNAandRnomics陈润生2013-05-10 The17December2010issueofScienceincludesspecialsectionshighlightingtheBreakthrough of the YearandInsights of the Decade.InsightsoftheDecadeShiningaLightontheGenomesDarkMatterThescopeofthis“darkgenome”becameapparentin2001,whenthehumangenomewasfirstpublis
2、hed.Scientistsexpectedtofindasmanyas100,000genespackedintothe3billionbasesofhumanDNA;theywerestartledtolearnthattherewerefewerthan35,000.(Thecurrentcountis21,000.)Protein-codingregionsaccountedforjust1.5%ofthegenome.CouldtherestofourDNAreallyjustbejunk?非编码RNA与RNA组学研究的源起一、基因组研究Noncodingsequences:Sequ
3、encesingenome,whicharenotcodingforanyproteins.Howmanypersentofthehumangenomearenoncodingsequences?Morethan97%!Proportionoffunctionalelementswithingenomes二、转录组研究基因组和转录Genome and transcription (tiling array data)编码蛋白序列Protein coding sequence人基因组的2-3%线虫 基因组的25%基因组的转录水平Transcriptional activity人 基因组的90%(
4、40-50X)线虫 基因组的70%(2-3X)绝大部分的转录产物是非编码RNAThe majority of transcripts are non-coding RNAs物种间最主要的差别也是非编码RNAThe major differences among different organisms are ncRNAs基因组的转录情况Transcriptional output/complexityTheEncyclopediaofDNAElements(ENCODE)Consortiumisaninternationalcollaborationofresearchgroupsfunded
5、bytheNationalHumanGenomeResearchInstitute(NHGRI).ThegoalofENCODEistobuildacomprehensivepartslistoffunctionalelementsinthehumangenome,includingelementsthatactattheproteinandRNAlevels,andregulatoryelementsthatcontrolcellsandcircumstancesinwhichageneisactive.5 September 2012 - ENCODE results published
6、in Nature, Science and other journalsTheresultsoftheENCODEprojectwerepublishedtodayinacoordinatedsetof30paperspublishedinmultiplejournals.Thesepublicationsaretheresultofcross-consortiumintegrativeanalysis,coveringmorethan4millionregulatoryregionsinthehumangenomemappedaspartofENCODE.Thecoordinatedpub
7、licationsetincludesonemainintegrativepaperandfiveotherpapersinthejournalNature;18papersinGenome Research;andsixpapersinGenome Biology.TheENCODEdataaresocomplexthatthethreejournalshavedevelopedapioneeringwaytopresenttheinformationinanintegratedformtheytermthreads.Sincethesametopicswereaddressedindiff
8、erentwaysindifferentpapers,the NatureENCODEwebsitewasdevelopedtoallowreaderstofollowatopicthroughallofthepapersintheENCODEpublicationset.Inadditiontothesepublications,sixreviewarticlesarebeingpublishedintheJournal of Biological Chemistry,andotheraffiliatedpapersinScience,Cell,andotherjournals.Thenew
9、IntegrativeAnalysispageonthisportalprovideslinksanddescriptivematerialforthesepublicationsandrelatedanalysisresources.三、非编码RNA的功能研究X-inactivation is the mammalian dosage compensation mechanism, used to equalize X-linked gene dosage between male and female cells. In mammals dosage compensation occurs
10、 by the transcriptional silencing of one X chromosome in all female somatic cells. The silencing of the inactive X chromosome is achieved by an X chromosome wide alteration in chromatin structure, from active euchromatin to inactive heterochromatin. X-inactivation is nucleated and bidirectionally pr
11、opagated from the X-inactivation center. The Xist gene lies within the X-inactivation center and is required to initiate X chromosome inactivation. Xist encodes a large, spliced, polyadenylated, noncoding RNA that is expressed exclusively from the otherwise inactive X chromosome. Cytologically Xist
12、RNA appears as a cluster of particles that coat the inactive X chromosome. Spread of Xist RNA correlates temporally with the spread of silencing along the X chromosome. The two most interesting questions that arise from this characterization of Xist are: how does Xist RNA function in altering chroma
13、tin structure and how is Xist itself regulated? http:/www.ucsf.edu/pibs/faculty/panning.htmlBarbara Panning Lab SmallRNAandRNAInterference(RNAi） A number of recent reports have shown that small RNA molecules play significant roles in both gene silencing and regulation of developmental timing. Small,
14、 processed double-stranded RNAs (also called short interfering RNAs or siRNAs) mediate the phenomenon known as RNA interference (RNAi) post-transcriptional gene silencing (PTGS) induced by the introduction of dsRNA and have biological roles in viral resistance in plants and transposon silencing in C
15、aenorhabditis elegans. Another group of small RNA molecules, known as small temporal RNAs (stRNAs), regulates C. elegans developmental timing through translational repression of target transcripts.InRNAi,dsRNAintroducedintosusceptibleorganismsisprocessedinto22nucleotide(nt)siRNAs.These22ntsiRNAssubs
16、equentlybindtothehomologousregionoftheirtargettranscriptandtagitfornucleasecleavage.ThusgenesilencingiseffectedbydestructionofthetargetmRNA.A novel class of small RNAs bind to MILI protein in mouse testesNATURE|Vol 442|13 July 2006 Small RNAs bound to Argonaute proteins recognize partially or fully
17、complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteinsknown as the Piwi familyis required for germ- and stem-cell development in invertebrates, and two Piwi membersMILI and MIWIare essential for spermatogenesis in mouse. Here we describe a new cl
18、ass of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The small RNAs are 2631 nucleotides (nt) in lengthclearly distinct from the 2123 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)and we refer to them as Piwi-interacting RNAs or pi
19、RNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalia
20、n spermatogenesis.http:/www.prl.msu.edu/PLANTncRNAs/ We have collected existing data on plant noncoding RNAs and expanded on this by examining about 20,000 Arabidopsis ESTs for characteristics of noncoding RNAs. About 15 putative Arabidopsis ncRNAs have been reported in the literature or have been a
21、nnotated. Several have homologs in other plants, but all appear to be plant-specific with the exception of SRP RNA. Conversely, none of about 30 ncRNAs reported from yeast, bacteria or animal systems have homologs in Arabidopsis. Noncoding RNA in PlantsPrionsborrowRNANature16OCT2003Theinfectiousagen
22、tsofpriondiseasesuchasCreutzfeldtJakobdiseasearethoughttobecomposedofprotein,withnoassociatednucleicacids.Butthatmaynotbetheendofthestory.Anexperimentinacell-freeamplificationsystemshowsthatunidentifiedhostRNAmoleculesarerequiredforefficientconversionofnormalprionproteinintoitspathogenicform.Interes
23、tingly,theseRNAmoleculesarenotpresentininvertebratespecies.Thispointstoapossibleinvolvementofhost-codedRNAinthepathogenesisofpriondiseases,andalsoprovidesasimplewayofincreasingthesensitivityofdiagnostictestsbasedonthePMCA(proteinmisfoldingcyclicamplification)method.Some examples about noncoding Some
24、 examples about noncoding RNAs and diseases RNAs and diseases ElevatedexpressionofPCGEM1,aprostate-specificgenewithcellgrowth-promotingfunction,isassociatedwithhigh-riskprostatecancerpatientsGyorgy Petrovics*,1, Wei Zhang2, Mazen Makarem1, Jesse P Street1, Roger Connelly1, Leon Sun1, Isabell A Seste
25、rhenn2, Vasantha Srikantan1, Judd W Moul1,3 and Shiv Srivastava1Oncogene (2004) 23, 605611PCGEM1 is a novel, highly prostate tissue-specific,androgen-regulated gene. Here, we demonstrate thatPCGEM1 expression is significantly higher in prostate cancer (CaP) cells of African-American men than in Cauc
26、asian-American men.PCGEM1 PCGEM1 appears to be a noncoding functional RNAappears to be a noncoding functional RNAgene (Srikantan gene (Srikantan et alet al., 2000)., 2000).His-1: A noncoding RNA implicated in mouse leukemogenesisFan Xu, Molly McFarland and David S. Askew*Histol. Histopathol., 1999,
27、14, 235241. The His-1 gene is highly conserved among vertebrate species and is transcribed as a single spliced and polyadenylated cytoplasmic RNA that shares several features in common with the emerging class of untranslated RNAs. A role for the His-1 gene in neoplastic transformation was first indi
28、cated by the identification of transcriptionally activated His-1 genes in a series of mouse leukemias, and more recent studies with antisense His-1 RNAs suggest that His-1 is involved in an oncogenic pathway that controls cell cycle progression. MALAT-1MALAT-1, , a novel noncoding RNA, and thymosin
29、a novel noncoding RNA, and thymosin b4 predict metastasisb4 predict metastasis and survival in early-and survival in early-stage non-small cell lung cancerstage non-small cell lung cancerPing Ji1,5, Sven Diederichs1,5, Wenbing Wang1, Sebastian Bo ing1, Ralf Metzger2, Paul M Schneider2, Nicola Tidow3
30、, Burkhard Brandt3, Horst Buerger4, Etmar Bulk1, Michael Thomas1,Wolfgang E Berdel1, Hubert Serve*,1 and Carsten Mu ller-Tidow*,1Oncogene (2003) 22, 8031Oncogene (2003) 22, 803180418041EvidenceforevolutionarilyconservedsecondarystructureintheH19 tumorsuppressorRNAVeronicaJuan,ChadCrainandCharlesWils
31、onNucleic Acids Research 2000 Vol. 28 1221-1227BREAKTHROUGH OF THE YEAR(2001):Science celebrates nine other areas in which important findings were reported this year, from subatomic to atmospheric and beyond.First runner-up: RNA ascending.ShortRNAsclearlyplayimportantbiologicalroles.Dozensofthemolec
32、ulesarenowknowntoexistinthenematodeandfruitfly.ThecodingforthesemoleculesiscontainedintheDNAsequence.Some100ofthesetinyRNAgeneshavebeenfoundinthegutbacteriumEscherichia coli,andsome200wereuncoveredinDNAfrommousebraintissue.Inthenematodeandfruitfly,theyseemtobeinvolvedindevelopment;inE. coli,theymayf
33、acilitaterapidresponsestoenvironmentalchangeandcouldservesimilarfunctionsinmammals.Nature391,806-811(19February1998)Potentandspecificgeneticinterferencebydouble-strandedRNAinCaenorhabditis elegansANDREWFIRE*, SIQUNXU*, MARYK.MONTGOMERY*, STEVENA.KOSTAS*, SAMUELE.DRIVER & CRAIGC.MELLO *Carnegie Insti
34、tution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Maryland 21210, USABiology Graduate Program, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USAProgram in Molecular Medicine, Department of Cell Biology, University of Massachuset
35、ts Cancer Center, Two Biotech Suite 213, 373 Plantation Street, Worcester, Massachusetts 01605, USA前景与值得关注的方向一、一、非编码RNA的主要部分是长的500nt长链非编码RNA(lncRNA)是一类转录本长度超过200nt的RNA分子，它们并不编码蛋白，而是以RNA的形式在多种层面上（表观遗传调控、转录调控以及转录后调控等）调控基因的表达水平。lncRNA起初被认为是基因组转录的“噪音”，是RNA聚合酶II转录的副产物，不具有生物学功能。然而，近年来的研究表明，lncRNA参与了X染色体沉默
36、，基因组印记以及染色质修饰，转录激活，转录干扰，核内运输等多种重要的调控过程，lncRNA的这些调控作用也开始引起人们广泛的关注。哺乳动物基因组序列中约4%9%的序列产生的转录本是lncRNA（相应的蛋白编码RNA的比例是1%），虽然近年来关于lncRNA的研究进展迅猛，但是绝大部分的lncRNA的功能仍然是不清楚的。许多lncRNA都具有保守的二级结构，剪切形式以及亚细胞定位，这种保守性和特异性表明它们是具有功能的。 Many small RNAs, such as microRNAs or snoRNAs, exhibit strong conservation across divers
37、e species (Bentwich 2005). In contrast, in general long ncRNAs lack strong conservation, which is often cited as evidence of non-functionality (Brosius 2005; Struhl 2007). However, many well-described long ncRNAs, such as Air and Xist, are poorly conserved (Nesterova 2001), suggesting that ncRNAs ma
38、y be subject to different selection pressures (Pang 2006).Bentwich I, Avniel A, Karov Y, et al. (July 2005). Identification of hundreds of conserved and nonconserved human microRNAs. Nature Genetics 37 (7): 76670. doi:10.1038/ng1590. PMID 15965474. Brosius J (May 2005). Waste not, want not-transcrip
39、t excess in multicellular eukaryotes. Trends in Genetics 21 (5): 2878. doi:10.1016/j.tig.2005.02.014. PMID 15851065. Nesterova TB, Barton SC, Surani MA, Brockdorff N (July 2001). Loss of Xist imprinting in diploid parthenogenetic preimplantation embryos. Developmental Biology 235 (2): 34350. doi:10.
40、1006/dbio.2001.0295. PMID 11437441. Poly-adenylated RNA versus total RNA transcriptionConserved versus not conservedH19, a long recognized ncRNA, shows significant sequence conservation and is known to control IGF2 on the opposite paternal chromosome by epigenetic control mechanisms (Feil et al., 19
41、94; Juan et al., 2000).Feil R., Walter J., Allen N. D., Reik W. (1994). Developmental control of allelic methylation in the imprinted mouse Igf2 and H19 genes. Development120, 29332943.Juan V., Crain C., Wilson C. (2000). Evidence for evolutionarily conserved secondary structure in the H19 tumor sup
42、pressor RNA. Nucleic Acids Res.28, 12211227. doi: 10.1093/nar/28.5.1221. XIST shows almost no sequence conservation between species yet consistently silences one X chromosome in females (Hendrich et al., 1993; Panning et al., 1997). Hendrich B. D., Brown C. J., Willard H. F. (1993). Evolutionary con
43、servation of possible functional domains of the human and murine XIST genes. Hum. Mol. Genet.2, 663672. doi: 10.1093/hmg/2.6.663.Panning B., Dausman J., Jaenisch R. (1997). X chromosome inactivation is mediated by Xist RNA stabilization. Cell90, 907916. doi: 10.1016/S0092-8674(00)80355-4. Large scal
44、e sequencing of cDNA libraries and more recently transcriptomic sequencing by next generation sequencing indicate that long noncoding RNAs number in the order of tens of thousands in mammals. However, despite accumulating evidence suggesting that the majority of these are likely to be functional, on
45、ly a relatively small proportion has been demonstrated to be biologically relevant. As of December 2012, 127 LncRNAs have been functionally annotated in LncRNAdb (a database of literature described LncRNAs).4Amaral,P.P.;Clark,M.B.;Gascoigne,D.K.;Dinger,M.E.;Mattick,J.S.(2010).LncRNAdb:Areferencedata
46、baseforlongnoncodingRNAs.Nucleic Acids Research39(Databaseissue):D146D151.doi:10.1093/nar/gkq1138.PMC3013714.PMID21112873.长、短NcRNA可能具有不同的结构与功能microRNA碱基匹配长NcRNA折叠形成空间结构Non-Coding RNA Has Role in Inherited Neurological Disorder, and Maybe Other Brain Diseases TooResearchers have discovered that expre
47、ssion of the ataxin-7 gene - the cause of the neurological disorder spinocerebellar ataxia type 7 - has two regulators: a highly conserved, multi-tasking protein called CTCF and, surprisingly, an adjacent promoter containing non-coding RNA. (Credit: Illustration courtesy of ChristinaTakamatsuButler,
48、 UC San Diego, publishedintheJune22issueofthejournalNeuron.)ALargeIntergenicNoncodingRNA Inducedbyp53MediatesGlobalGeneRepressioninthep53ResponseCell 142, 409419, August 6, 2010在这篇文章，作者发现P53可以直接提高lincRNA-P21的表达，然后lincRNA-P21与蛋白hnRNP-K结合，再调节其他基因的表达（如图）。transcriptional regulation For example, the ncRN
49、A Evf-2 functions as a co-activator for the homeobox transcription factor Dlx2, which plays important roles in forebrain development and neurogenesis (Feng 2006; Panganiban 2002). Sonic hedgehog induces transcription of Evf-2 from an ultra-conserved element located between the Dlx5 and Dlx6 genes du
50、ring forebrain development (Feng 2006). Evf-2 then recruits the Dlx2 transcription factor to the same ultra-conserved element whereby Dlx2 subsequently induces expression of Dlx5. The existence of other similar ultra- or highly conserved elements within the mammalian genome that are both transcribed
51、 and fulfil enhancer functions suggest Evf-2 may be illustrative of a generalised mechanism that tightly regulates important developmental genes with complex expression patterns during vertebrate growth (Pennacchio 2006; Visel 2008).Feng J, Bi C, Clark BS, Mady R, Shah P, Kohtz JD (June 2006). The E
52、vf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes & Development 20 (11): 147084. doi:10.1101/gad.1416106. PMC 1475760. PMID 16705037. The formation of RNA duplexes between complementary ncRNA and mRNA may mask key ele
53、ments within the mRNA required to bind trans-acting factors, potentially effecting any step in post-transcriptional gene expression including pre-mRNA processing and splicing, transport, translation, and degradation.The splicing of mRNA can induce its translation and functionally diversify the reper
54、toire of proteins it encodes. The Zeb2 mRNA, which has a particularly long 5UTR, requires the retention of a 5UTR intron that contains an internal ribosome entry site for efficient translation (Beltran 2008). However, retention of the intron is dependent on the expression of an antisense transcript
55、that complements the intronic 5 splice site (Beltran 2008). Therefore, the ectopic expression of the antisense transcript represses splicing and induces translation of the Zeb2 mRNA during mesenchymal development.Post-transcriptional regulationIndeed it was recently shown that BC1 is associated with
56、 translational repression in dendrites to control the efficiency of dopamine D2 receptor-mediated transmission in the striatum (Centonze 2007) and BC1 RNA-deleted mice exhibit behavioural changes with reduced exploration and increased anxiety (Lewejohann 2004).Centonze D, Rossi S, Napoli I, et al. (
57、August 2007). The brain cytoplasmic RNA BC1 regulates dopamine D2 receptor-mediated transmission in the striatum. The Journal of Neuroscience 27 (33): 888592. doi:10.1523/JNEUROSCI.0548-07.2007. PMID 17699670. Lewejohann L, Skryabin BV, Sachser N, et al. (September 2004). Role of a neuronal small no
58、n-messenger RNA: behavioural alterations in BC1 RNA-deleted mice. Behavioural Brain Research 154 (1): 27389. doi:10.1016/j.bbr.2004.02.015. PMID 15302134. translational regulation Epigenetic modifications, including histone and DNA methylation, histone acetylation and sumoylation, affect many aspect
59、s of chromosomal biology, primarily including regulation of large numbers of genes by remodeling broad chromatin domains. While it has been known for some time that RNA is an integral component of chromatin, it is only recently that we are beginning to appreciate the means by which RNA is involved i
60、n pathways of chromatin modification (Chen 2008; Rinn 2007; Sanchez-Elsner 2006)Chen X, Xu H, Yuan P, et al. (June 2008). Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133 (6): 110617. doi:10.1016/j.cell.2008.04.043. PMID 18555785. Rin
61、n JL, Kertesz M, Wang JK, et al. (June 2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129 (7): 131123. doi:10.1016/j.cell.2007.05.022. PMC 2084369. PMID 17604720. Sanchez-Elsner T, Gou D, Kremmer E, Sauer F (February 2006). Noncoding RN
62、As of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax. Science 311 (5764): 111823. Bibcode:2006Sci.311.1118S. doi:10.1126/science.1117705. PMID 16497925. epigenetic regulation ImprintingMany emergent themes of ncRNA-directed chromatin modification were first apparent within the
63、phenomenon of imprinting, whereby only one allele of a gene is expressed from either the maternal or the paternal chromosome. In general, imprinted genes are clustered together on chromosomes, suggesting the imprinting mechanism acts upon local chromosome domains rather than individual genes. These
64、clusters are also often associated with long ncRNAs whose expression is correlated with the repression of the linked protein-coding gene on the same allele. Indeed, detailed analysis has revealed a crucial role for the ncRNAs Kcnqot1 and Igf2r/Air in directing imprinting (Braidotti 2004). Almost all
65、 the genes at the Kcnq1 loci are maternally inherited, except the paternally expressed antisense ncRNA Kcnqot1 (Mitsuya 1999).Braidotti G, Baubec T, Pauler F, et al. (2004). The Air noncoding RNA: an imprinted cis-silencing transcript. Cold Spring Harbor Symposia on Quantitative Biology 69: 5566. do
66、i:10.1101/sqb.2004.69.55. PMID 16117633. Mitsuya K, Meguro M, Lee MP, et al. (July 1999). LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Human Molecular Genetics 8 (7): 120917. doi:10.1093/hmg
67、/8.7.1209. PMID 10369866. Telomeres have been long considered transcriptionally inert DNA-protein complexes until it was recently shown that telomeric repeats may be transcribed as telomeric RNAs (TelRNAs) (Schoeftner 2008) or telomeric repeat-containing RNAs (Azzalin 2007). These ncRNAs are heterog
68、eneous in length, transcribed from several sub-telomeric loci and physically localise to telomeres. Their association with chromatin, which suggests an involvement in regulating telomere specific heterochromatin modifications, is repressed by SMG proteins that protect chromosome ends from telomere l
69、oss (Azzalin 2007). In addition, TelRNAs block telomerase activity in vitro and may therefore regulate telomerase activity (Schoeftner 2008).Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J (November 2007). Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromo
70、some ends. Science 318 (5851): 798801. Bibcode:2007Sci.318.798A. doi:10.1126/science.1147182. PMID 17916692. Schoeftner S, Blasco MA (February 2008). Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nature Cell Biology 10 (2): 22836. doi:10.1038/ncb1
71、685. PMID 18157120. Telomeric non-coding RNAsFigure 1. Paradigms for how long ncRNAs function. Recent studies have identified a variety of regulatory paradigms for how long ncRNAs function, many of which are highlighted here. Transcription from an upstream noncoding promoter (orange) can negatively
72、(1) or positively (2) affect expression of the downstream gene (blue) by inhibiting RNA polymerase II recruitment or inducing chromatin remodeling, respectively. (3) An antisense transcript (purple) is able to hybridize to the overlapping sense transcript (blue) and block recognition of the splice s
73、ites by the spliceosome, thus resulting in an alternatively spliced transcript. (4) Alternatively, hybridization of the sense and antisense transcripts can allow Dicer to generate endogenous siRNAs. By binding to specific protein partners, a noncoding transcript (green) can modulate the activity of
74、the protein (5), serve as a structural component that allows a larger RNAproteincomplex to form (6), or alter where the protein localizes in the cell (7). (8) Long ncRNAs (pink) can be processed to yield small RNAs, such as miRNAs, piRNAs, and other less well-characterized classes of small transcrip
75、ts.Long noncoding RNAs: functional surprises from the RNA worldGenes Dev. 2009 23: 1494-1504Access the most recent version at doi:10.1101/gad.1800909 作用于非编码基因上游启动子区（桔色）通过干扰RNA多聚酶II的招募或影响染色体重塑,抑制(1)或增强(2)下游基因（蓝色）的表达。(3)、反义RNA（紫色）转录本通过与有互补序列的蛋白编码基因转录本形成互补双链，进而干扰mRNA的剪切，从而产生不同的剪切形式。(4)、正义与反义RNA转录本形成的互补
76、双链，在Dicer酶作用下产生内源性的siRNA。长非编码RNA(lncRNA)转录本（绿色）可以:(5)、通过结合到特定蛋白质上，调节该蛋白的活性;(6)、作为结构组分与蛋白质形成核酸蛋白质复合体;(7)、通过结合到特定蛋白上，改变该蛋白的亚细胞定位。(8)、长非编码RNA(lncRNA)转录本（粉红色）作为小分子RNA，如miRNA，piRNA或其他未知的小分子RNA的前体。A ncRNA transcribed from the HOX locus, HOTAIR, has been shown to bind to the polycomb repressor complex 2 (P
77、RC2) and affect expression of over 300 coding gene targets as part of a general reprogramming of breast cancer cells from a locally aggressive epithelial phenotype to an invasive and metastatic “mesenchymal” phenotype (Gupta et al., 2010).Gupta R. A., Shah N., Wang K. C., Kim J., Horlings H. M., Won
78、g D. J., Tsai M. C., Hung T., Argani P., Rinn J. L., Wang Y., Brzoska P., Kong B., Li R., West R. B., van de Vijver M. J., Sukumar S., Chang H. Y. (2010). Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 10711076. doi: 10.1038/nature08975.1、Found100nove
79、lnoncodingRNAsandtheirgenesinC.elegans 通过建立新的实验方案，在C.elegans基因组中发现了100个迄今国际上未见报导的全新的非编码RNA基因。这些基因不同于已知的各种类的NcRNA，也不同于microRNA。这是新类型的RNA基因。论文已发表在 Genome ResearchGenome Research 16: 20-29, 2006; 16: 20-29, 2006; NCBI accession number: AY948555- AY948719NCBI accession number: AY948555- AY948719 The ste
80、m-bulge RNAs of C. elegans 发现两个新非编码发现两个新非编码RNA基因家族基因家族(Toclassifytwonewcategories)The snRNA like RNAs of C. elegans 发现了三个独特的非编码基因上游发现了三个独特的非编码基因上游motif (UM1-3) (confirm three special upstream motifs of noncoding genes)located within 40-80 bp upstream of the transcription initiation sites of the ncRN
81、A loci were further revealed by MEME (Bailey and Elkan, 1995). The expression levels of non-motif snoRNAs with the frequencies of ESTs corresponding to exons of their host genes, produced a distinct positive correlation not found for motif-containing loci.提出了非编码基因是一个独立的系统并给出了其基提出了非编码基因是一个独立的系统并给出了其基
82、本调控模式本调控模式(foundthatmanyofthencRNAgenesarelocatedintheintronsofhostprotein-codinggenesandareunderthecontrolofindependentpromoterelements.)Analysis of transcription levels of 106 ncRNA families were carried out with Northern blot. 61 showed variation exceeding two standard variation, composed of 6 di
83、stinct expression clusters. Developmentally regulated ncRNAsPublic release date: 9-Jan-2006Contact:MariaSmitsmitcshl.edu516-422-4013ColdSpringHarborLaboratoryPregnantprotein-codinggenescarryRNAbabiesScientists characterize large numbers of independently expressed, non-protein-coding RNA genes in the
84、 introns of protein-coding genesBEIJING, ChinaScientistsfromtheChineseAcademyofScienceshaveperformedacomprehensiveanalysisofsmall,non-protein-codingRNAsinthemodelnematode,C. elegans.Theycharacterize100heretofore-undescribedtranscripts,includingtwonovelclasses;theyprovideinsightsintothegenomicstructu
85、reandtranscriptionalregulationofnon-codingRNAs;andtheyunderscoretheimportanceofnon-codingRNAsinnematodedevelopment.TheirworkappearsthismonthinthejournalGenome Research.*Thesignificanceofnon-protein-codingRNAsascentralcomponentsofvariouscellularprocesseshasrisensharplyovertherecentyears,explainsProf.
86、RunshengChen,principalinvestigatoronthestudy.ExcludingmicroRNAs(miRNAs),orsmalltranscriptsthathaverecentlyreceivedwidespreadattentionandareknowntoplayimportantrolesintranscriptionalregulation,smallnon-codingRNAs(orncRNAs)inC. eleganshavenotbeenextensivelyinvestigateduntilnow.Usinganew,high-throughpu
87、tproceduretoclonesmall,full-lengthncRNAs,Chenslaboratoryisolatedandcharacterized161uniquetranscripts.AmajoradvantageofthenewcloningprocedureisthatitachievesanextraordinarilyhighdetectionrateforncRNAsbycurrentstandards.Studiespublishedoverrecentyearshaveonlybeenabletoreachadetectionrateofabout3%,buto
88、urmethodreachedadetectionrateof30%a10-foldincreaseincloningefficiency,explainsChen.ItslikegoingfromaModelTFordtoaFerrariinonefellswoop!Ofthe161transcriptsdetectedbyChensgroup,100werenoveland61werepreviouslyknownorpredicted.Amongthe100novelgenes,30hadnoknownfunction,whereas70belongedtotheubiquitouscl
89、assofsmallnucleolarRNAs(snoRNAs).Basedonsequenceandstructuralfeatures,Chenandhiscolleagueswereabletoclassifymorethanhalfofthe30unknownRNAsintotwonewcategories:stem-bulgeRNAs(sbRNAs)andsmallnuclear-likeRNAs(snlRNAs).Bothclassesoftranscriptsexhibitedenhancedexpressionduringthelaterstagesofwormdevelopm
90、ent,indicatingafunctionalroleforthesetranscriptsindevelopmentalprocesses.TheinterestingthingaboutnematodesisthattheirgenomicorganizationofbothsnoRNAsandotherncRNAsisquitedifferentfromotheranimals,saysChen.Incontrasttothegenomesofothermetazoans,wheremostsnoRNAsarefoundinintronsandareunderthecontrolof
91、independentpromoters,nematodesnoRNAlociarebothintergenicandintronic(withandwithoutpromoters).Interestingly,plantsnoRNAsareprimarilylocatedinintergenicregions.OtherncRNAgenes(i.e.,non-snoRNAgenes)aremainlylocatedinintergenicregionsinbothplantsandanimals.Butinnematodes,Chensteamfoundthatmanyoftheseoth
92、erncRNAgenesarelocatedintheintronsofhostprotein-codinggenesandareunderthecontrolofindependentpromoterelements.Finally,Chenandhiscolleaguesestimatedthat2700ncRNAgenesarepresentintheC. elegansgenome.Oneparticularlyintriguingaspectofthenon-codingtranscriptomeisitspotentialtofilltheregulatorygapcreatedb
93、ythesurprisinglylownumberofprotein-codinggenesinhigherorganisms,saysChen.Betweenone-celledyeast,thousand-cellednematodes,andtrillion-celledmammals,thereisadifferenceofamere6,000to19,000to25,000inprotein-codinggenenumbers.Wethinkthatregulationbynon-codingRNAaccountsforthisdiscrepancyandhelpstoexplain
94、theadditionalbiologicalcomplexityofhigherorganisms.现在我们在C.elegant已有1400多个新中长NcRNA!在Humen已有近900个新长NcRNA!2、构建了NcRNA数据库(have built the noncoding RNA databaseNONCODE) 收集了在各种杂志上发表的、网站上公布的所有被实验证实的NcRNA基因，发展了相应的软件及检索工具，建成了NcRNA数据库。相关论文已送Nucleic Acids Research。韩国已要求成为我们的镜象。上网仅两个多月点击我们数据库的目前已超过12万次（平均每天约2000
95、次）来自约60，000个不同的IP地址。论文已发表在2005年第一期Nucleic Acids Research Nucleic Acids Research 上。上。上。上。新版本已发表在2007年Nucleic Acids Research Nucleic Acids Research 上。上。上。上。ncRNA数据库二、RNA是生物网络的元件Nature415,141-147(2002)3、ncRNA网络和双色网络 Afraction/portionofthemRNA-like-ncRNAsserveasvectorsorstorageformsforshortncRNAs,whicha
96、rereleasedwhenneededtoperformtheircellularfunctions.PriMiridentified84likelypre-miRNAcandidates,correspondingto80individualme-ncRNAs. MicroRNA-EncodingLongNoncodingRNAs(ME-ncRNA)BMC Genomics 2008,9:236 MicroRNAsregulatemicroRNAsanetworkofmutualmicroRNAcontrolTrans in Genetics2008,24:3231、Messenger-l
97、ikencRNAsshowmiRNA-relatedreductionsinexpression,thatmeanstheywouldberegulatedbymicroRNAs(miRNAs)likemRNAtargets.2、ThemRNA-like-ncRNAsserveasvectorsorstorageformsforshortncRNAs（MicroRNA). AndtheyhypothesizedaregulatoryrelationshipbetweenmicroRNAs.Ifthismodelistrue,itwillfurthergreatlystimulatethecom
98、munitysinterestintheregulatoryroleofmicroRNA,andrepresentsamilestoneinexploringmicroRNAsfunctionality.三、NcRNA调节的多样性miRNA-inducedtranscriptionalinhibitionWang Y,PatelDJ.Nature.2008,456:209-213； Nature.2008,456:921-926；Nature.2009,461:754-761； Nat Struct Mol Biol.2010,17：781-787.miRNA-inducedtranscrip
99、tionalgenesilencingPromotorasscoiatedRNAmiR-10ainhibitshoxd4transcriptionTransfectionstudiesHoxd4expressionisinhibitedbymiR-10aAnti-miR-10aincreaseshoxd4 expressionInhibitionnotmediatedbythe3UTRNuclearrun-onassayconfirmstranscriptionalinhibitionTranscriptionalactivityinthepromoter regionofatargetgen
100、eisrequiredforsiRNA-inducedtranscriptionalsilencingPNAS 2007 miRNA不仅具有负调控作用，也可以激活基因的表达。Vasudevan实验室发现,在细胞周期过程中,miRNA效应在抑制作用和活化作用间摆动.在静态细胞中(G0期),miRNA活化翻译和上调基因表达,而在其他细胞循环/增殖期则继续发挥抑制作用.miRNA激活作用与富含腺嘌呤/尿嘧啶元件(adenylate/uridylate-richelements,AREs)相关.ARE是miRNA活化翻译的信号,在miRNA指导下,miRISC复合物成员如Ago,FXRP被招募到ARE
101、上,激活翻译、上调基因表达.ARE元件是一种mRNA不稳定元件,位于mRNA3UTR，对很多mRNA来说，它都是保守的。 VasudevanS,TongY,SteitzJA.Switchingfromrepressiontoactivation:microRNAscanup-regulatetranslation.Science,2007,318(5858):19311934.其中一个相互作用的过程并不依赖MicroRNA的seedsequence，并且会激发CEBPAmRNA的释放。这些结果表明，microRNA具有控制细胞命运的双重路径和功效，它不仅通过mRNA来发挥作用，还通过调节蛋白来
102、发挥作用。 Cell,Volume140,652-665,5March2010四、NcRNA是与疾病紧密相关的Examples of lncRNAs correlated with diseases/disorders（ Kannanganattu V. Prasanth and David L. Spector， Genes Dev. 2007 21: 11-42）NcRNAs Disease/disorder ReferenceNcRNAs with altered expression levels in cancerAntisense intronic Prostate cancer
103、Reis et al. 2004ncRNAs BC1 Overexpressed in several cancers Chen et al. 1997b BC200 Overexpressed in breast, cervix, esophagus, lung, Chen et al. 1997a; Iacoangeli et al. 2004 ovary, parotid, and tongue cancer（乳腺、宫颈、食道、肺、卵巢、腮腺、舌癌）BCMS B-cell neoplasia （B-细胞瘤） Wolf et al. 2001CMPD Campomyelic displas
104、ia （短指發育不良） Ninomiya et al. 1996DD3 Overexpressed in prostate cancer Bussemakers et al. 1999H19 Overexpressed in liver and breast cancer Looijenga et al. 1997; Lottin et al. 2002HIS-1 Overexpressed in myeloid leukemia Askew et al. 1994HOST2 Expressed in ovarian cancer cells Rangel et al. 2003MALAT-1
105、 NSCLC, endometrial sarcoma, and hepatocellular Ji et al. 2003; Lin et al. 2006; Yamada et al. carcinoma （子宫内膜肉瘤、肝细胞癌） 2006NC612 Prostate cancer A.P. Silva et al. 2003NCRMS Elevated expression in alveolar rhabdomyosarcoma （横纹肌肉瘤） Chan et al. 2002OCC1 Overexpressed in colon carcinoma Pibouin et al. 2
106、002PCGEM1 Overexpressed in prostate cancer Srikantan et al. 2000PEG8/IGF2AS Fetal tumors Okutsu et al. 2000SRA Steroid receptor activated RNA isoform expressed Lanz et al. 1999 in breast cancerTRNG10 Various cancers Roberts et al. 1998U50HG snoRNA host gene; located at the chromosomal Tanaka et al.
107、2000 breakpoint involved in human B-cell lymphomaNcRNAs correlated with neurological diseases/disordersBC200 Alzheimers Lukiw et al. 1992DISC2 Schizophrenia and bipolar affective disorder Millar et al. 2000, 2004; Blackwood et al.2001IPW Prader-Willi syndrome Wevrick et al. 1994Prion-associated RNAs
108、 Prion pathologies Deleault et al. 2003; Supattapone 2004PSZA11q14 Reduced expression in brains of patients with Polesskaya et al. 2003 schizophrenia （精神分裂症） RAY1/ST7 Autistic disorder Vincent et al. 2002SCA8 (KLHL1 antisense) Spinocerebellar ataxia type 8 Nemes et al. 2000; Mutsuddi et al. 2004UBE3
109、A-AS Angelman syndrome Chamberlain and Brannan 2001ZNF127AS Prader-Willi syndrome Jong et al. 1999 NcRNAs correlated with other diseases/disorders22k48 HIRA intronic transcript deleted in DiGeorge Pizzuti et al. 1999 syndromeC6orf37OS Antisense transcript from C6orf37 locus within Matsuzaka et al. 2
110、002 diffuse panbronchiolitis critical regionCOPG2IT1 Russell-Silver syndrome Yamasaki et al. 2000DGCR5 Disrupted in DiGeorge syndrome Sutherland et al. 1996H19 Beckwith-Wiedemann syndrome Sparago et al. 2004LIT1 Beckwith-Wiedemann syndrome Niemitz et al. 2004LIT1 Romano-Ward, Jervell and Lange-Niels
111、en Horike et al.2000 syndromesMESTIT 1 Russell-Silver syndrome T. Li et al. 2002; Nakabayashi et al. 2002PRINS Psoriasis Sonkoly et al. 2005此外： 一种名为HOTAIR的lincRNA在原发乳腺癌和其迁移过程中表达量上升。 HOTAIR表达水平可以作为最终迁移和死亡预测指标。上皮细胞癌中HOTAIR表达上升，导致PRC2重新定位且类似于胚胎成纤维细胞，致使组蛋白H3的27位赖氨酸甲基化、基因表达改变，癌症的侵入和迁移增加。相反，HOTAIR缺失可以抑制癌症
112、的迁移，特别是在有过量PRC2的细胞中。这些发现表明lincRNA在调控癌症表观组时有积极作用，可能作为癌症诊断和治疗的重要靶点。（Chang, Howard Y. Nature Volume 464 April 2010）Cancer-Associated Long Non-Coding RNA Regulates Pre-mRNA SplicingScienceDaily (Sep. 27, 2010) Researchers report this month that MALAT1, a long non-coding RNA that is implicated in certain
113、 cancers, regulates pre-mRNA splicing - a critical step in the earliest stage of protein production. Their study appears in the journal Molecular Cell. 已有的研究结果表明，在高等生物中，小分子非编码RNA在干细胞干性维持、胚胎发育、细胞分化、凋亡、代谢、信号传导、感染以及免疫应答等几乎所有重要生命活动中发挥关键的调控作用，提示生物体内可能存在着由RNA介导的遗传信息表达调控网络。JBC（周琪、王秀杰）：发现指示干细胞发（周琪、王秀杰）：发现指示干细胞发育潜能的标记育潜能的标记77 Thank you!
