1、基因组“暗物质”环状RNA2014.10.08Group 111953年，克里克和沃森提出中心法则年，克里克和沃森提出中心法则唯一以法则命名的生物学理论。唯一以法则命名的生物学理论。核酶核酶1989年诺贝尔化学奖年诺贝尔化学奖 美国科学家切赫美国科学家切赫 加拿大科学家奥尔特曼加拿大科学家奥尔特曼2RNAi2006年诺贝尔医学奖 安德鲁安德鲁法尔法尔 克雷格克雷格梅洛梅洛 3 几乎所有的RNA都是线性的，为数不多的关于植物和动物中的环状RNAs的记述。遗传意外 实验人为因素4环形RNA分子20世纪70年拟病毒也称为类类病毒，它是一种环状单链RNA。它的侵染对象是植物病毒。绒毛烟、苜蓿、茛菪、地
2、下三叶草51.1979 年，Hsu 和Coca-Prados利用电子显微镜第一次观察到RNA 可以以环状的形式存在于真核细胞的细胞质中。2.Arnberg 等在酵母的线粒体中又发现了circRNA3.1993 年，人们在人体细胞的转录本中也发现了一些由外显子构成的circRNA。外显子转录本发生错误剪接而形成的低丰度外显子转录本发生错误剪接而形成的低丰度RNA 分子分子？6Jeck 等在人类成纤维细胞中检测出了高达25 000 多种的circRNA；Memczak 等通过RNA-seq 鉴定出1950 种人类circRNA、1903 种小鼠circRNA(其中81 种与人类circRNA 相同
3、)和724 种线虫circRNA。线性线性RNAs的优势的优势有可能一直是假象假象。经典的RNA测序方法具有特征性分子“尾巴“的那些分子。环状RNAs的末端连接在一起，缺乏这些尾巴，因此被普遍忽略掉。7 2012年，斯坦福大学医学院的分子生物学家年，斯坦福大学医学院的分子生物学家Julia Salzman和同事们和同事们他们报道称在寻找用传统方法可能忽视的RNA过程中，发现了过量的环状人类RNAs，并进行了研究。2013年，年，Rajewsky研究小组研究小组环状RNA充当分子“海绵”，结合并封闭了称作microRNAs的微小基因调控子。并且在斑马鱼中表达这一环状RNA或敲除miR-7可以改变
4、大脑发育。2013年，丹麦奥胡斯大学的年，丹麦奥胡斯大学的Thomas Hansen和和Jorgen Kjems1500个核苷酸构成的一个环状大RNA上包含了70个miR-7的结合位点。MicroRNAs是一些通过结合和阻止mRNA翻译阻断基因表达的短片段RNA。2014年，年，William R Jeck1 和和Norman E Sharpless系统检测、识别circRNA的方法，全新的对环形RNA进行研究的方法与认识。（Detecting and characterizing circular RNAs）8 circRNAs与正常RNAs竞争，机体还以牺牲正常RNA为代价来生成它们。在大
5、脑中环状RNA分子以高水平生成，在许多情况下来自具有非常重要功能的一些基因。circRNAs在脑功能以及有可能在脑疾病中脑疾病中发挥重要作用，与阿尔兹海默阿尔兹海默发生相关。muscleblind可以促进和调控一组环状RNAs的生成,表明circRNAs有可能参与了营养不良性肌强直营养不良性肌强直的形成。?927 February 2013Natural RNA circles function as efficient microRNA spongesThomas B.Hansen,Trine I.Jensen,Bettina H.Clausen,Jesper B.Bramsen,Bente
6、 Finsen,+et al.Nature 495,384-388 doi:10.1038/nature1199327 February 2013Circular RNAs are a large class of animal RNAs with regulatory potencySebastian Memczak,Marvin Jens,Antigoni Elefsinioti,Francesca Torti,Janna Krueger,+et al.Nature 495,333-338 doi:10.1038/nature1192827 February 2013Molecular b
7、iology:Circles reshape the RNA worldKenneth S.KosikNature 495,322-324 doi:10.1038/nature11956Rajewsky研究小组研究小组在斑马鱼中表达这一环状RNA或敲除miR-7可以改变大脑发育。环状RNA充当分子“海绵”，结合并封闭了称作microRNAs的微小基因调控子。101112To identify circRNAs,designed an algorithmPrevious methodA new algorithmOnly used existing exon-intron annotations
8、Filter out reads that aligned contiguously to the genome,retaining the spliced readsDid not explicitly identify the splice sites used for circularizationMap the terminal parts of each candidate read independently to the genome to find unique anchor positionsAssumed that each pair of mates in paired-
9、end sequencing derives from the same RNA molecule.Anchor alignments can be extended such that the orginal read sequence aligns completelyThe inferred breakpoint is flanked by GU/AG splice signal13To identify circRNAs,designed an algorithmA new algorithmFilter out reads that aligned contiguously to t
10、he genome,retaining the spliced readsMap the terminal parts of each candidate read independently to the genome to find unique anchor positionsAnchor alignments can be extended such that the orginal read sequence aligns completelyThe inferred breakpoint is flanked by GU/AG splice signal14Identify tha
11、t circRNAs are not just rare and specifically expressed15Sequence conservation within circRNAs16qPCR test circRNA17Digest with RNase R181924h after blocking transcription circRNAs were highly stable,exceeding the stability of the housekeeping gene GAPDH.2021circRNAs were identified and its character
12、izationNot rareSpecifically(spatial and temporal)CDS exonsConservationRNase R resistantstable22Can circular RNA be a miRNA sponge?23The circRNA CDR1as is bound by the miRNA protein AGO24The circRNA CDR1as is cytoplasmic and highly expressed每个细胞中CDR1as可能最多结合20,000个miR-7分子蓝色，种子匹配；暗红，AGOPAR-CLIP阅读25 si
13、RNA depletion of CDR1as induces repression of miR-7 target genes26CDR1as and miR-7 have overlapping and specific expression in neuronal tissues27Knockdown of miR-7 expression of CDR1as causes midbrain defects28Expression of CDR1as causes midbrain defects29 Knockdown of miR-7 or expression of CDR1as
14、causes midbrain defects30ABOUT AUTHORSSebastian Memczak Nikolaus Rajewsky computational analysis of animal small RNA deep sequencing data focusing on the identification of miRNAs and their target genes computationally detect functional small peptides(so called“micro peptides”)in fliesScientific Head
15、 of the Berlin Institute for Medical Systems Biology31Systems Biology of Gene Regulatory Elements,Max-Delbru ck-Center for Molecular Medicine,Robert-Ro ssle-Strasse 10,13125 Berlin,Germany The Rajewsky lab combines theoretical/computational and experimental methods to understand more about gene regu
16、lation in animalsA major focus is on post-transcriptional gene regulation by small RNAs(for example microRNAs)and RNA binding proteins32DISCUSSIONCDR1as can act as a post-transcriptional regulator by binding miR-7 in brain tissues:CDR1as is densely bound by miRNA effector molecules CDR1as harbours 7
17、4 miR-7 seed matches,often deeply conserved CDR1as is expressed highly,stably and mostly cytoplasmic CDR1as and miR-7 share specific expression domains in mouse embryonic brain human/mouse CDR1as is circularized in vivo and is not detectable as a linear molecule human/mouse CDR1as sequences,when inj
18、ected into zebrafish,and miR-7 knock down have similar phenotypes in brain 33Future studies should elucidate how CDR1as can be converted into a linear molecule and targeted for degradationCDR1as miR-671miR-7PAK1/FAK134The phenotype induced by CDR1as expression in zebrafish was only partially rescued
19、 by expressing miR-7,indicating that CDR1as could have functions beyond sequestering miR-7 bind in trans 3/UTRs of target mRNAsmiR-7 binds CDR1as assembly of larger complexes of RNA or protein 35How many other circRNAs exist：certainly much larger a few tissues/developmental stages with stingent cuto
20、ffs circular RNAs in fibroblasts were described 36circRNAs probably compete with other RNAs for miRNA binding circRNA from the SRY locus has seed sites for murine miRNAs 37It is appealing to speculate that occasional circu-larization of exons is easy to evolve and may provide a mechanism for rapid e
21、volution of stably and well expressed regulatory RNAs 38There is no reason to think that circRNAs function predominantly to binding miRNAsseed matches for viral miRNAs within human circRNAs RBPs miRNA sponges in bacteria39Supplementary Reading4041High-throughput sequencing(RNA-seq)libraries prepared
22、 from ribosome-depleted RNAIdentified 25,000 distinct RNA species in human fibroblasts that contained noncolinear exons(a“backsplice”)reproducibly enriched by exonuclease degradation of linear RNA.circular RNA(ecircRNA),rather than linear RNACircular RNA(ecircRNA)more stable than associated linear m
23、RNAs in vivo;the abundance of circular molecules exceeded that of associated linear mRNA by 10-foldecircRNAs were not bound to ribosomesdegraded by siRNAs,may act as competing endogenous RNAs.Application of this method to murine testis RNA identified69 ecircRNAs in precisely orthologous locations to
24、 human circular RNAs.paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and miceThat ecircRNAs are abundant,stable,conserved and nonrandom products of RNA splicing that could be involved in control of gene expressioncontrol of gene expression.Abstract&
25、IntroductionAbstract&Introduction42Purpose:Biochemical enrichment of nonlinear RNAs detection of more rare and diverse circular RNA forms.Hs68 cells+RNase R Preference:Continuous mappingSpliced mappings Fusion mappings Treatment with RNase R Decreased coverage of linear products;Enrichment of reads
26、from exons included in circular products&increased reads mapping as backsplice junctionsUnbiased identification of RNA circlesUnbiased identification of RNA circles43Mapping artifactsNonsequential exons harbored in linear products(resulting from either RNA trans-splicing or cleavage of ecircRNAs)Enr
27、ichment of circular RNAs by CircleSeqEnrichment of circular RNAs by CircleSeq44Single exon ecircRNAs：The intervening exons not directly part of the backsplice also showed enrichment by RNase RIntrons are spliced from most circular formsMultiexon circles：The intervening exons not directly part of the
28、 backsplice also showed enrichment by RNase RIntrons are spliced from most circular formsThat backsplice-containing transcripts identified by this method are diverse,generally RNase R-resistant and include most previously described circular RNAsCircular RNAs contain predominantly Circular RNAs conta
29、in predominantly exonic sequenceexonic sequence45Abundence：The relative rate ranging from3200%,Introns are spliced from most circular forms.Indicate:The formation of circular RNAs is considerable.The formation of circular RNAs is considerable.Rare ecircRNAs arising from pervasive background levels o
30、f RNA circularization(occasional error in splicing).46Novel backsplice eventsANRIL 14-5;ASXL1;FOXN2;HIPK3;KIAAO182;LPRAR1;MYO9B;ZFY Enrichment Linear RNASTBP,GAPDH,18S Decreased circular RNA:a subset of exonsTrans-spliced products:repeated exons(longer than full-length)Oligo-dT priming significantly
31、 reduced TBP and GAPDHs levels of backspliced productsthese species were not polyadenylatedCharacters：(1)contain backsplices,(2)are enriched by RNase R treatment,(3)are not polyadenylated,and(4)are of smaller size than linear mRNAs emanating from thesame locusEcircRNAs resulting from cis-rather than
32、 trans-splicing.EcircRNAs resulting from cis-rather than trans-splicing.Circular RNA Circular RNA validationvalidation47Abundence：The relative rate ranging from3200%,Introns are spliced from most circular forms.Indicate:The formation of circular RNAs is considerable.The formation of circular RNAs is
33、 considerable.Conservation of circular RNA production in paralogous genes orthologous genes IndicatesEvolutionary preservation of circular RNA formation.Conservation of abundant circularized transcriptsConservation of abundant circularized transcripts51Representative ecircRNAs UntranslatedUntranslat
34、ed Targeted by siRNATargeted by siRNAEcircRNAs can be targeted by RNA interference.Circular RNAs might regulateregulate transcription through an effect on microRNA bindingRepresentative ecircRNAs are untranslated Representative ecircRNAs are untranslated but can be targeted by siRNAbut can be target
35、ed by siRNA52Method:Treated with actinomycin DMeasurement：Half-lifeData sets:the circular RNA isoforms were highly stable (with transcript half-lives exceeding 48 h)Associated linear transcripts exhibited half-lives of 1Results Enrichment of protein kinases and related proteins among the set of gene
36、s producing ecircRNAs (protein kinase transcripts exhibit abundant circularization)No specific subfamily of kinase was particularly associated with ecircRNA production (disproportionately associated with kinase expression)54(*)P 105,(*)P 1010,(*)P Noncomplementary.Upstream and downstream introns flanking circularized exons tended to be large(on average more than approximately threefold longer than introns flanking control exons,(P 1015)Bioinformatic analyses of ecircRNAsBioinformatic analyses of ecircRNAs55个人观点供参考，欢迎讨论
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