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1、Transcriptomics,转录组学,Transcriptome:An evolving definition,(The population of)mRNAs expressed by a genome at any given time(Abbott,1999)The complete collection of transcribed elements of the genome.(Affymetrix,2004),Transcribed elements,mRNAs:35,913 transcripts(including alternative spliced variants)
2、Non-coding RNAstRNAs(497 genes)rRNAs(243 genes)snmRNAs(small non-messenger RNAs)microRNAs and siRNAs(small interferring RNAs)snoRNAs(small nucleolar RNAs)snRNAs(small nuclear RNAs)Pseudogenes(2,000),Transcriptomics,Definition The study of characteristics and regulation of the functional RNA transcri
3、pt population of a cell/s or organism at a specific time.Scopethe population of functional RNA transcripts.the mechanisms that regulate the production of RNA transcriptsdynamics of the trancriptome(time,cell type,genotype,external stimuli),一、转录组学研究全部RNA的表达及功能,转录组（transcriptome）指特定状态下一种细胞或组织所能转录出来的所有
4、RNA的总和。包括编码RNA，即mRNA和非编码RNA(non-coding RNA,ncRNA)转录组学（transcriptomics）：是在整体水平上研究细胞基因转录情况及转录调控规律的科学。RNA组学（RNomics）：是分析、鉴定非信使小RNA（small non-messenger RNA,snmRNA）在特定状态下表达情况、功能及其与蛋白质的相互作用。,转录组的特点：受到内外多种因素的调节，因而是动态可变的。能够揭示不同物种、不同个体、不同细胞、不同发育阶段及不同生理病理状态下的基因差异表达信息。,Observing the transcriptome,Focussed Expe
5、rimental Approaches:Northern Blotting AnalysisRT-PCR(quantitative or semi-quantitative)High throughput Approaches:Closed System Profiling:Microarray expression profiling Open System Profiling:Serial analysis of gene expression(SAGE)Massively Parallel Signature Sequencing(MPSS),微阵列（microarray）SAGEMPS
6、S,研究技术,（一）微阵列是大规模基因组表达谱研究的主要技术,大规模表达谱或全景式表达谱（global expression profile）：是生物体（组织、细胞）在某一状态下基因表达的整体状况。微阵列或基因芯片（DNA chip）:利用光导化学合成、照相平板印刷以及固相表面化学合成等技术，在固相表面合成成千上万个寡核苷酸探针，并与放射性同位素或荧光物标记的来自不同细胞、组织或整个器官的DNA或mRNA反转录生成的第一链cDNA进行杂交，然后用特殊的检测系统对每个杂交点进行定量分析。,Experimental overview:,Limit of Detection:1 in 30,000
7、transcripts 20 transcripts/cell,Red increase of Cy5 sample transcriptsGreen increase of Cy3 sample transcriptsYellow equal abundance,Affymetrix GeneChip,Limits:1:100,000 transcripts 5 transcripts/cell,http:/,Affymetrix:,Gene Expression ArraysTranscripts/GenesArabidopsis Genome24,000 C.elegans Genome
8、 22,500 Drosophila Genome 18,500 E.coli Genome 20,366 Human Genome U133 Plus47,000 Mouse Genome39,000 Yeast Genome5,841(S.cerevisiae)&5,031(S.pombe)Rat Genome30,000 Zebrafish14,900 Plasmodium/Anopheles 4,300(P.falciparum)&14,900(A.gambiae)Barley(25,500),Soybean(37,500+23,300 pathogen),Grape(15,700)C
9、anine(21,700),Bovine(23,000)B.subtilis(5,000),S.aureus(3,300 ORFS),Xenopus(14,400),Microarray and GeneChip Approaches,Advantages:RapidMethod and data analysis well described and supportedRobustConvenient for directed and focussed studiesDisadvantages:Closed system approachDifficult to correlate with
10、 absolute transcript numberSensitive to alternative splicing ambiguities,（二）SAGE在转录物水平研究细胞或组织基因表达模式,SAGE的基本原理：利用锚定酶（anchoring enzyme，AE）和位标酶（tagging enzyme，TE）切割DNA分子的特定位置（一般近3端），分离SAGE标签（长约14 bp，可藉此鉴定基因组中的所有基因），并将这些标签串联起来，然后对其进行测序特点：可全面提供生物体基因表达谱信息可用来定量比较不同状态下组织或细胞的所有差异表达基因,Anchoring Enzyme NlaIII,
11、recognition site:The 3 terminus of adaptor A and Bare both TCCRACTAG,where a recognition site of Tagging Enzyme MmeI flanked with NlaIII,Hu M,Polyak K.Nature Protocols 2006,Tagging Enzyme MmeI recognition site:,Hu M,Polyak K.Nature Protocols 2006,SAGE,Advantages:Potential open system method new tran
12、scripts can be identifiedAccuracy of unambiguous transcript observationDigital output of dataQuantitative and qualitative informationDisadvantages:Characterising novel transcripts is often computationally difficult from short tag sequencesTag specificity(recently increased length to 21 bp)Length of
13、tags can vary(TE enzyme activity variable with temperature)A subset of transcripts do not contain enzyme recognition sequenceSensitive to a subset of alternative splice variants,（三）MPSS是以基因测序为基础的基因表达谱分析新技术,MPSS的原理：一个含有能够特异识别转录子的信息标签序列（1020 bp）与长的连续分子连接在一起，测出mRNA的一端包含一个10至20个碱基的标签序列。每一标签序列在样品中的频率（拷贝数
14、）代表了与该标签序列相应的基因表达水平。基因表达水平是以计算mRNA拷贝数为基础，是一个数字表达系统。只要将病理和对照样品分别进行测定，即可进行严格的统计检验，能测定表达水平较低、差异较小的基因，而且不必预先知道基因的序列。,四、RNA组学研究全部snmRNA,人类基因组序列特点：2万2.5万个基因，与蛋白质合成有关的序列占整个基因组的2%左右，其余98%的基因组序列没有得到注释。RNA组学研究范畴：小分子RNA，包括 snRNA、snoRNA、scRNA、siRNA、miRNA,Small RNA Catalogues Naqvi(2009)Int J Biol Sci,Within cel
15、ls there are a variety of discovered small RNAs in length in 19-30 nt,recently governing diverse cellular processes such as development,differentiation across the eukaryotic kingdom.siRNA:short interfering RNA as defensive mechanism to protect host genome integrity from intrusion of foreign nucleic
16、acids.miRNA(microRNA):21-30 nt in length transcribed from host genome loci,involved in wide cellular processes,specific to development and differentiation.tasiRNA(trans-acting short interfering RNA):21 nt length taken endogenous transcript as template,under RdRP activity,followed by Dicer to produce
17、 tasiRNA.In human and fly without tasiRNA it is due to absent to RdRP.,27,Small RNAs Catalogues,rasiRNA(repeat-associated RNA):24-26 nt long products of DCL3(Dicer like protein,in plant)on long dsRNA formed,usual in retro-transposon loci with methylation,to play role in gametogenesis via recruiting
18、chromatin remodeling proteins to modulate chromatin status.scnRNA(small scanning RNA):29 nt long scnRNA,reported from protozoan,derived from micro-nuclei,eliminated original loci of genome,given birth to macro-nuclei.lsiRNA(long siRNA):30-40 nt in length induced in response to bacterial infection or
19、 growth condition.piRNA(piwi-acting RNA),21-U RNA,28,MicroRNA 简介,(1)长度为21nt左右核苷酸的内源性单链小分子RNA；(2)存在65nt左右的发夹结构前体；(3)基因座位于蛋白质基因间隔区；(4)其DNA序列在近源物种间高度保守。miRNA具有十分重要的调控功能，它们主要参与基因转录后水平的调控。能够通过与靶mRNA特异性的碱基配对引起靶mRNA的降解（植物中较为常见）或者抑制其翻译（动物中较为常见），从而影响了靶mRNA的表达。目前发现miRNA是一个庞大的小分子调控RNA家族，广泛存在于各种动植物中，参与细胞增殖和分化、细
20、胞凋亡、胚胎发育、形态建成以及疾病发生等一系列重要的生命过程。最近发现一系列与肿瘤发生相关的和人类病毒编码的miRNA，揭示miRNA在哺乳动物基因表达调控中具有重要作用。,30,Rana(2007)J Cell Physiol,*Small RNA biogenesis-RISC formation-RNAi&Its Mechanism-Cell Phenotypy*,31,32,Rana(2007)Nature Rev Mol Cell Biol,33,*Overview on RNA Interference,Genomic loci transcribed by RNA Pol II,
21、III and IV to form double-stranded RNAs(dsRNA)or viral RNA dependent RNA polymerase(RdRP)to generate dsRNA.DsRNA trimmed by RNase III(Drosha in nuclear and Dicer in cytoplasm)to small duplex RNA with 19-30 bp.A single stranded RNA unwound by Argonaute as guide RNA and loaded into RISC(RNA induced si
22、lencing complex).Complementary to target RNA by guide RNA in RISC and triggered RNA interference.,RNA InterferenceTarget RNA to be degradation.Target RNA to be translational repression or destabilization.Target RNA to be transcriptional repression.The genomic locus of target RNA to become heterochro
23、matin or degradation.,34,*Biogenesis of miRNAs and siRNAsBartel(2004)Cell,36,*Biogenesis of miRNAs and siRNAsBartel(2004)Cell,Structure loci:genomic segment mirror repeat.Convergent transcription:two promoters to locate each sides of one segment,simultaneous transcription.One version of transposon t
24、o align inversely to the other.Bidirectional transcription:two promoters to locate each side of the gene.Trans-interaction:gene forward-transcript(sense transcript)to interact with pseudogene reverse-transcript(antisense transcript).Pseudogene duplication and orientation opposite.RdRP to direct dsRN
25、A generation in plants,fungi and C elegans,virus,not in human.,37,38,Biogeneses of siRNA,miRNA and piRNA,and Their Pathways Zamore(2009)Nat Rev Genet,39,Annotation Fig:siRNAi Pathway,40,RNA dependent RNA polymerase(RdRP)activity on aberrant transcripts or transcript having full or partial complement
26、arity.These are recognized and processed by nuclear Dicer(different from the one involved in microRNA pathway)and this siRNA-Dicer complex is then exported to cytoplasm.The siRNA-Dicer complex then recruits Argonaute that unwind the duplex to form si-RISC/RITS.Transcripts bearing complementary seque
27、nces to guide siRNA strand are cleaved by RNase activity of Argonaute2.To confer immunity,siRNAs-Dicer complex may also traffic in systemic fashion that is achieved by Systemic RNA Interference-Defective(SID-1;in animals)/Phloem Small RNA binding protein-1(PSRP1;in plants).The exogenous siRNA pathwa
28、y follows parallel to endogenous pathway,but differs in the fact that the cytoplasmic Dicer generates the siRNA duplexes.The RITS complex lead to transcriptional gene silencing that involves various proteins.,Naqvi(2009)Int J Biol Sci,41,miRNAi Pathway Naqvi(2009)Int J Biol Sci,Annotation Fig miRNAi
29、 Pathway,42,After being transcribed,the pri-miRNAs stem-loop structure is acted upon by Drosha(that also confers to miRNA strand and target specificity)and generates pre-miRNA.Sometimes,these precursors are edited by Adenosine Deaminase Acting on RNA(ADARs)at specific positions(generally+4 and+44)ch
30、anging adenine to inosine.In plants,the DCL1 generates miR duplex in the nucleus that is methylated at terminal bases by HEN1.These are then transported to cytoplasm with the assistance of Exportin-5/HASTY.From here,Dicer comes into play(in animals)and generates miRNA duplexes that will be incorpora
31、ted into micro Ribo-Nucleo-Protein(mi-RNP)complex.After the removal of passenger strand mature miRNA then guides the functional protein complex to the targets.In mammals,miRNAs bearing nuclear signal sequences can traffic back to the nucleus.Depending upon the proteins associated with miRNA leads to
32、 either cleavage of target mRNA or modulate the translation turnover by(g)translation activation or repression of respective mRNAs.The repressed mRNAs are transferred to structures called P-bodies.,43,44,Annotation Fig:siRNA mediated genomic DNA elimination,45,Production of double stranded RNA(thin
33、lines)by bidirectional transcription of genomic DNA.Production of scnRNAs by dicer-like protein Dcl1p(yellow);Association of scnRNAs with Argonaute Twi1p(green)in the cytoplasm;Scanning in the parental macronucleus.The RNA helicase Ema1p(red)is required for the association of Twi1p with non-coding R
34、NA;Heterochromatin formation and IES elimination in the developing new macronucleus.(histone methylation:purple;chromodomain proteins:dark green;hypothetical excisase:orange;IES:internal elimination sequence,repeat-seq or transposon-like).,46,47,Long&Small ncRNAs to Trigger Transcriptional Repressio
35、n via Methylation of DNA&Histones Morris(2009)Epigenetics,48,Long antisense non-coding RNAs expressed at bidirectionally transcribed genes.The sense and antisense ncRNA strands could pair each other forming 2nd structured ncRNAs,i.e.RNA duplex.Those 2nd structured ncRNAs interact with particular sit
36、es in the promoter of sense strand at the bidirectionally transcribed gene and also influence the recruitment of Ago-1,DNMT3a and HDAC-1 to this target site.Small synthetic antisense ncRNAs can be designed to take advantage of the endogenous mechanism and also utilize the same pathway to transcripti
37、onally silence gene expression.The end result of either small or long antisense ncRNA transcription silencing is the targeted epigenetic remodeling of the particular RNA targeted genome loci.,49,siRNA-RISC Suppression on Protein Synthesis Carthew RW(2009)Cell,50,miRNAs Patterns Vs Cell Types in Tiss
38、ues&Cancers Lee(2008)RNA,Pearson Coefficient(miR-1,9,137)miRNAs Pattern-Specificity in cancer cell lines(37 cell lines).miRNAs Pattern-Specificity in normal tissues(22 tissues).miRNAs Pattern-Specificity with normal liver Vs liver cancer.miRNAs Pattern-Specificity with normal pancreas Vs pancreas ca
39、ncer.,miRNA Cancer Cell Lines(37)-Pattern(copy/cell),51,Heterochronic phenotypes of some C.elegans mutants.Two representative cell lineages,V and P,show the transformations caused by the absence(0)or continuous activity(gf)of two heterochronic genes,lin-4 and lin-14.V cells normally divide twice in
40、the L2 stage(arrowhead)and differentiate at the end of the L4 stage(arrow),but these events occur one stage early in a precocious mutant and not at all in the retarded mutants.P cells show a completely different overall pattern from the V lineage,but their fates are likewise changed in the heterochr
41、onic mutants in this case,through alteration of cell-cycle length(gray bar).,miRNA Vs Development:Lin14,or Lin4 Vs Heterochronic Phenotypes of C.elegans Mutants Moss(2007)Cell,52,miRNA Vs Cellular Differentiation Moss(2007)Cell,53,The microRNA-Target Paradigm Moss(2007)Cell,MicroRNAs increase in abu
42、ndance at each stage and repress specific targets that encode developmental regulators.The change in regulators at each stage leads to a succession of developmental events.This has been a useful paradigm for understanding the heterochronic gene pathway of C.elegans.But it should be noted that it is
43、an oversimplification and does not account for some key features of the pathway.,54,*MicroRNAs and Cellular Phenotypy Kosik(2010)Cell,Annotation Fig:miRNAs Vs Cellular Phenotypy Kosik(2010)Cell,55,The cell state is the complete list of constituent molecules within a cell each at a specific number of
44、 copies at one particular moment in time,and at its unique location?The levels of all transcripts are one component of the cell state and each transcript is expressed at a range of levels with some maxima and minima depicted as boundaries.Within these boundaries the cell maintains a discrete identit
45、y,for example a specific type of differentiated cell.When a cell changes its identityfor example by reprogramming to a stem cell or undergoing malignant transformationnew boundaries are established for the transcriptome.Transcription factors drive cells across boundaries to new identities and operate in feed-back and feed-forward loops with microRNAs(miRNAs).miRNA profiles reflect cell identity with very high accuracy and therefore reduce high-dimensional cell state values to a single profile.,
