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1、,非编码RNA与RNA组学Noncoding RNA and Rnomics陈润生 2013-05-10,The 17 December 2010 issue of Science includes special sections highlighting the Breakthrough of the Year and Insights of the Decade.Insights of the DecadeShining a Light on the Genomes Dark Matter,The scope of this“dark genome”became apparent in
2、2001,when the human genome was first published.Scientists expected to find as many as 100,000 genes packed into the 3 billion bases of human DNA;they were startled to learn that there were fewer than 35,000.(The current count is 21,000.)Protein-coding regions accounted for just 1.5%of the genome.Cou
3、ld the rest of our DNA really just be junk?,非编码RNA与RNA组学研究的源起,一、基因组研究,Noncoding sequences:Sequences in genome,which are not coding for any proteins.How many persent of the human genome are noncoding sequences?More than 97%!,Proportion of functional elements within genomes,二、转录组研究,基因组和转录 Genome and t
4、ranscription(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%(40-50X)线虫 基因组的 70%(2-3X)绝大部分的转录产物是 非编码RNA The majority of transcripts are non-coding RNAs物种间最主要的差别也是 非编码RNA The major differences among different organisms are ncRNAs,基因组的转录
5、情况 Transcriptional output/complexity,The Encyclopedia of DNA Elements(ENCODE)Consortium is an international collaboration of research groups funded by the National Human Genome Research Institute(NHGRI).The goal of ENCODE is to build a comprehensive parts list of functional elements in the human gen
6、ome,including elements that act at the protein and RNA levels,and regulatory elements that control cells and circumstances in which a gene is active.,5 September 2012-ENCODE results published in Nature,Science and other journalsThe results of the ENCODE project were published today in a coordinated
7、set of 30 papers published in multiple journals.These publications are the result of cross-consortium integrative analysis,covering more than 4 million regulatory regions in the human genome mapped as part of ENCODE.The coordinated publication set includes one main integrative paper and five other p
8、apers in the journal Nature;18 papers inGenome Research;and six papers in Genome Biology.The ENCODE data are so complex that the three journals have developed a pioneering way to present the information in an integrated form they term threads.Since the same topics were addressed in different ways in
9、 different papers,theNature ENCODE websitewas developed to allow readers to follow a topic through all of the papers in the ENCODE publication set.In addition to these publications,six review articles are being published in the Journal of Biological Chemistry,and other affiliated papers inScience,Ce
10、ll,and other journals.The newIntegrative Analysis page on this portal provides links and descriptive material for these publications and related analysis resources.,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 fema
11、le cells.In mammals dosage compensation occurs 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-inact
12、ivation is nucleated and bidirectionally propagated 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 inac
13、tive X chromosome.Cytologically Xist 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 Xi
14、st RNA function in altering chromatin structure and how is Xist itself regulated?http:/www.ucsf.edu/pibs/faculty/panning.htmlBarbara Panning Lab,Small RNA and RNA Interference(RNAi）,A number of recent reports have shown that small RNA molecules play significant roles in both gene silencing and regul
15、ation of developmental timing.Small,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
16、and transposon silencing in Caenorhabditis elegans.Another group of small RNA molecules,known as small temporal RNAs(stRNAs),regulates C.elegans developmental timing through translational repression of target transcripts.In RNAi,dsRNA introduced into susceptible organisms is processed into 22 nucleo
17、tide(nt)siRNAs.These 22 nt siRNAs subsequently bind to the homologous region of their target transcript and tag it for nuclease cleavage.Thus gene silencing is effected by destruction of the target mRNA.,A novel class of small RNAs bind to MILI protein in mouse testesNATURE|Vol 442|13 July 2006 Smal
18、l RNAs bound to Argonaute proteins recognize partially or fully 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 esse
19、ntial for spermatogenesis in mouse.Here we describe a new class 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(siRNA
20、s)and we refer to them as Piwi-interacting RNAs or piRNAs.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 t
21、he molecular function of Piwi proteins in mammalian 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 h
22、ave been reported in the literature or have been annotated.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 Plants,P
23、rions borrow RNANature 16OCT 2003,The infectious agents of prion disease such as CreutzfeldtJakob disease are thought to be composed of protein,with no associated nucleic acids.But that may not be the end of the story.An experiment in a cell-free amplification system shows that unidentified host RNA
24、 molecules are required for efficient conversion of normal prion protein into its pathogenic form.Interestingly,these RNA molecules are not present in invertebrate species.This points to a possible involvement of host-coded RNA in the pathogenesis of prion diseases,and also provides a simple way of
25、increasing the sensitivity of diagnostic tests based on the PMCA(protein misfolding cyclic amplification)method.,Some examples about noncoding RNAs and diseases,Elevated expression of PCGEM1,a prostate-specific gene with cell growth-promoting function,is associated with high-risk prostate cancer pat
26、ientsGyorgy Petrovics*,1,Wei Zhang2,Mazen Makarem1,Jesse P Street1,Roger Connelly1,Leon Sun1,Isabell A Sesterhenn2,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 ex
27、pression is significantly higher in prostate cancer(CaP)cells of African-American men than in Caucasian-American men.PCGEM1 appears to be a noncoding functional RNAgene(Srikantan et al.,2000).,His-1:A noncoding RNA implicated in mouse leukemogenesisFan Xu,Molly McFarland and David S.Askew*Histol.His
28、topathol.,1999,14,235241.,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 w
29、as first indicated 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-1,a novel noncoding RNA,and thymosin
30、 b4 predict metastasis and survival in early-stage non-small cell lung cancerPing Ji1,5,Sven Diederichs1,5,Wenbing Wang1,Sebastian Bo ing1,Ralf Metzger2,Paul M Schneider2,Nicola Tidow3,Burkhard Brandt3,Horst Buerger4,Etmar Bulk1,Michael Thomas1,Wolfgang E Berdel1,Hubert Serve*,1 and Carsten Mu ller-
31、Tidow*,1Oncogene(2003)22,80318041,Evidence for evolutionarily conserved secondary structure in the H19 tumor suppressor RNAVeronica Juan,Chad Crain and Charles WilsonNucleic Acids Research 2000 Vol.28 1221-1227,BREAKTHROUGH OF THE YEAR(2001):Science celebrates nine other areas in which important fin
32、dings were reported this year,from subatomic to atmospheric and beyond.First runner-up:RNA ascending.,Short RNAs clearly play important biological roles.Dozens of the molecules are now known to exist in the nematode and fruit fly.The coding for these molecules is contained in the DNA sequence.Some 1
33、00 of these tiny RNA genes have been found in the gut bacterium Escherichia coli,and some 200 were uncovered in DNA from mouse brain tissue.In the nematode and fruit fly,they seem to be involved in development;in E.coli,they may facilitate rapid responses to environmental change and could serve simi
34、lar functions in mammals.,Nature 391,806-811(19 February 1998)Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans ANDREWFIRE*,SIQUNXU*,MARYK.MONTGOMERY*,STEVENA.KOSTAS*,SAMUELE.DRIVER&CRAIGC.MELLO*Carnegie Institution of Washington,Department of Embryology,115 W
35、est 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 Massachusetts Cancer Center,Two Biotech Suite 213,373 Plantation Street,Wo
36、rcester,Massachusetts 01605,USA,前景与值得关注的方向,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，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
37、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 diverse species(Bentwich 2005).In contrast,in general long ncRNAs lack str
38、ong 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 may be subject to different selection pressures(Pang 2006).Bentwich I,Avniel A,Kar
39、ov 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-transcript excess in multicellular eukaryotes.Trends in Genetics 21(5):2878.doi:10.1016/j.tig.2005.02.01
40、4.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:10.1006/dbio.2001.0295.PMID 11437441.Poly-adenylated RNA versus total RNA transcriptionConserved versus not conse
41、rved,H19,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.,1994;Juan et al.,2000).Feil R.,Walter J.,Allen N.D.,Reik W.(1994).Developmental control of allelic methylation in the
42、 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 suppressor RNA.Nucleic Acids Res.28,12211227.doi:10.1093/nar/28.5.1221.,XIST shows almost no sequence conservation between species y
43、et 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 conservation 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.,
44、Jaenisch R.(1997).X chromosome inactivation is mediated by Xist RNA stabilization.Cell90,907916.doi:10.1016/S0092-8674(00)80355-4.,Large scale sequencing of cDNA libraries and more recently transcriptomic sequencing by next generation sequencing indicate that long noncoding RNAs number in the order
45、of tens of thousands in mammals.However,despite accumulating evidence suggesting that the majority of these are likely to be functional,only a relatively small proportion has been demonstrated to be biologically relevant.As of December 2012,127 LncRNAs have been functionally annotated in LncRNAdb(a
46、database of literature described LncRNAs).4 Amaral,P.P.;Clark,M.B.;Gascoigne,D.K.;Dinger,M.E.;Mattick,J.S.(2010).LncRNAdb:A reference database for long noncoding RNAs.Nucleic Acids Research 39(Database issue):D146D151.doi:10.1093/nar/gkq1138.PMC 3013714.PMID 21112873.,长、短NcRNA可能具有不同的结构与功能microRNA 碱基
47、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 Too,Researchers have discovered that expression of the ataxin-7 gene-the cause of the neurological disorder spinocerebellar ataxia type 7-has two regulators:a highly conserved,multi-tasking pr
48、otein called CTCF and,surprisingly,an adjacent promoter containing non-coding RNA.(Credit:Illustration courtesy of ChristinaTakamatsuButler,UC San Diego,published in the June 22 issue of the journal Neuron.),A Large Intergenic Noncoding RNA Induced by p53 Mediates Global Gene Repression in the p53 R
49、esponse Cell 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 ncRNA Evf-2 functions as a co-activator for the homeobox transcription factor Dlx2,which plays important roles in forebrain development and neu
50、rogenesis(Feng 2006;Panganiban 2002).Sonic hedgehog induces transcription of Evf-2 from an ultra-conserved element located between the Dlx5 and Dlx6 genes during forebrain development(Feng 2006).Evf-2 then recruits the Dlx2 transcription factor to the same ultra-conserved element whereby Dlx2 subseq
