2Main Contens Intruduction Regulation by RNAs in Bacteria Regulation by RNAs in Eukaryotes
3IntruductionWhat is regulatory RNA ?The discovery of regulatory RNAThe importance of regulatory RNA
4What is regulatory RNA ?regulatory RNAs are RNA molecules that function without being translated into proteins.Non-coding RNAs: Functional RNA molecules that are not translated into proteins .
5The Discovery of regulatory RNA Background:人类基因组草图带给科学家们的困惑The discovery of miRNAs
6Background:人类基因组草图带 给科学家们的困惑 Background:人类基因组草图带给科 学家们的困惑 1. Human genome: 3 billion chemical base pairs~20,500 genes是果蝇的2倍，啤酒酵母的4倍Human genome: only 1-2％ code for protein，24％ is the sequence of intron,what is another 74% for ?3. Human genome: 90% is transcribed.Human Genome Project information archive ( )
7Background:人类基因组草图带 给科学家们的困惑 The data from HGP Tells us: Most of the RNA transcribed from your genome doesn’t make protein.The complexity of higher organisms is not decided by the number of proteins coded by its genome.
8More and more scientists believe that Non-coding RNA functions in gene-regulatory networks that underlie the complexity of higher organisms.8
10The discovery of miRNAs Victor AmbrosGary RuvkunmiRNA was first discovered in 1993 by Victor Ambros at Harvard (lin-4)The second miRNA Let-7 was discovered in 2000 by Frank Slack as a postdoc at Harvard (Ruvkun lab)
11The first discovered miRNA lin-4 in 1993 Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell Jan 23;116 (2 Suppl):S93-6.Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell Jan 23;116 (2 Suppl):S89-92Thought to be an oddity not a general phenomenon
12Breakthrough with BlastN of the second miRNA (stRNA) let-7 Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P,Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature Nov 2;408(6808):86-9.
14The number of the identified miRNAs is growing rapidly in recent years. Over 5000 miRNAs have been found until the August of 2007 (The miRBase Sequence Database). These miRNAs are from primates, rodents, birds, fish, worms, flies, plants and viruses.The data are freely available to all through the web interface at and in flatfile form from ftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.
17microRNAs had been neglected for so many years because of their small size. The underlying reason is: people never dream that small RNAs will have important biological roles.
18The importance of regulatory RNA In the last few years scientists have been surprised by small nucleotide sequences, microRNAs and siRNAs (small interfering RNAs), that appear to play a role in both suppressing and promoting cancer.“We are at a transition in our understanding of RNA,” “RNA is taking on a new role. It is a regulatory molecule.” said Phillip A. Sharp, Ph.D., Nobel laureate and Institute Professor at the Massachusetts Institute of Technology.
19Regulatory RNA was first discovered in eukaryotes. Does Regulatory RNA exist in prokaryotes ?
20Regulation by RNAs in Bacteria Small RNAs (sRNA): regulation by base pairing.Riboswitches: regulation by metabolite-mediatedstructure changes3. Attenuation: Regulation by ribosome stop-mediatedformation of terminators
21Small RNAs (sRNA) Targets Regulation of translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively.MechanismRegulation by base pairing with the targeted sequences on mRNAs.Acts in trans similar to miRNA, but does not require specific machinery for action.
222. Riboswitches (核糖开关) Concept Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites et al to control gene transcription or translation.According to the factor thay sense, riboswitches can be divided into four types：Type小分子代谢物敏感型金属离子敏感型环境因素敏感型空载tRNA敏感型riboswitche
23 Metabolite-Binding Riboswitches TargetsRegulate translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectivelyMechanismReside upstream of the targeted mRNA, and form specific structure to bind its small molecule ligand.Act in cis by alteration of its own structure upon the binding of the small metabolites.
25Figure a. Control of transcription termination by a riboswitch 代谢物Figure b. Control of translation initiation by a riboswitch.代谢物
26The 2nd structures of 7 riboswitches and metabolites that they sense
273. Attenuation (衰减作用) target mechanism A premature transcription termination that switches off gene expression from amino acid biosynthetic operons after the corresponding amino acid is synthesized at an adequate level.mechanismRequires the participation of ribosomes that translate a leader peptide. The premature transcription termination is triggered by formation of an intrinsic terminator when ribosome read through codons of the amino acid that the operon synthesizes.
28The TRP operonThe trp operon encodes five structural genes required for tryptophan (色胺酸) synthesis.These genes are regulated to efficiently express only when tryptophan is limiting.Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation
29Fig 16-19Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of a leader RNA of 161 nt. (WHY?)
30The leader RNA and leader peptide of the trp operon
31Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的) Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的). Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA.The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites.Ribosome pause at these sites alter the secondary structure of the leader RNA, which eliminates the intrinsic terminator structure and allow the successful transcription of the trp operon.
32High Trp Low Trp Transcription of the leader RNA. RNA PolTranscription of the trp operon mRNA.
33Importance of attenuation A typical negative feed-back regulationUse of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan.Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation.Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.
34Regulation by RNA in Eukaryotes Molecular cell biology—Lesson 17(2)分子生物学Regulation by RNA inEukaryotes主讲：李 芬 教授/博士河南师范大学生命科学学院
35Main Contens Introduction The classification of non-coding RNA The origin, mechanism and function of regulatory RNAs in Eukaryotes
36Epigenetics Introduction Concept The study of meiotically and mitotically heritable changes in gene expression that are not coded for in the underlying DNA sequence.MechanismsDNA cytosine methylationhistone modificationsnon-coding RNAsHuman Genome Project information archive ( )
37What is non-coding RNA ? non-coding RNAs (ncRNAs) are RNA molecules that function withoutbeing translated into proteins.Non-coding RNAs: Functional RNA molecules that are not translated into proteins .
38Versatility in form and function non-coding RNA :Versatility in form and functionnon-coding RNA are Surprisingly numerous.non-coding RNA have very different functions.
39The importantce of non- coding RNA Human Genome sequnce:1-2% : coded for protein.90% is transcribed.24% : intron sequence.What is the left 74% for?Non-coding RNA functions in gene- regulatory networks that underlie the complexity of higher organisms.
40Housekeeping non-coding RNA The classification of non-coding RNAHousekeeping non-coding RNAmiRNAShort non-coding RNA(20-500nt)non-coding RNASiRNARegulatorynon-coding RNApiRNALong non-coding RNA(500-20,000nt)lncRNA
41The origin, mechanism and function of regulatory RNAs in Eukaryotes Topic 1. miRNA: micro-RNA (微 RNA )Topic 2. SiRNA：Small interfering RNA (小干扰 RNA )Topic 3. piRNA：Piwi interacting RNA ( piwi 偶联 RNA )Topic 4. lncRNA: long non-coding RNA (长非编码 RNA )
42Topic I. miRNA The definition and Origin of miRNA The biogenesis of miRNAThe action mode of miRNAMultiple Roles of miRNA
431 The definition and Origin of miRNA Size: ~21–23 nts Structure: short hairpin loopOrigin:Derived from cellular genemiRNAs are small ncRNAs of nucleotides, which leads to mRNA degradation or translational inhibition of its target genes. Encoded by eukaryotic nuclear DNA and involved in multiple cellular processes via base-pairing with complementary sequences within mRNA molecules.A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression. Encoded by eukaryoticnuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base-pairingwith complementary sequences within mRNA molecules.
44miRNA基因由Pol Ⅱ转录的300-1000 nts 初级转录产物 pri-miRNA The biogenesis of miRNA2miRNA的生成TranscriptionTranscriptionProcessingFunctional Complex assemblyProcessingFunctional Complex assemblyPol ⅡmiRNA genes are usually transcribed by RNA polymerase II (Pol II).The resulting transcript is capped with a specially modified nucleotide at the 5’ end, polyadenylated with multiple adenosines (a poly(A) tail),Pre-miRNA hairpins are exported out of the nucleus in a process involving the nucleocytoplasmic shuttler Exportin-5. In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer.miRNA基因由Pol Ⅱ转录的 nts 初级转录产物 pri-miRNA
453 The action mode of miRNA MiRNA binds to the target mNRA with limited complementarityMiRNA binds to the target mNRA with high complementarity. A given miRNA may have hundreds of different mRNA targets, and a given target might be regulated by multiple miRNAs.
46Multiple Roles of miRNA 4 (A) miRNA in developmentExpression of lin-4 allows： C. elegans to proceed to the late developmental stagemiRNAs are well conserved in both plants and animals, and are thought to be a vital and evolutionarily ancient component of genetic regulation.The first miRNA was discovered in the early 1990s. However, miRNAs were not recognized as a distinct class of biological regulators until the early 2000s. Since then, miRNA research has revealed different sets of miRNAs expressed in different cell types and tissues and has revealed multiple roles for miRNAs in plant and animal development and in many other biological processeslin-4 binds its target mRNAs by imperfect base pairing.Victor R. Ambros
48Multiple Roles of miRNA 4(C) miRNA controls the differentiation of thehematopoietic stem cell (调控造血干细胞分化)Three kinds of miRNA control the differentiation process of hematopoietic stem cell toward lymphocyte3种miRNA控制造血干细胞向淋巴细胞的分化过程( Science 2004)
49Multiple Roles of miRNA 4 (D) miRNA In cancer1. miRNA are cancer genes2. miRNA are cancer classifiers3. miRNA are cancer drivers4. miRNA are drugs and drug targetsBy measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type.
50Topic II. SiRNA The origin of SiRNA Biogenesis and regulation of SiRNA Three ways of SiRNA-guided gene silencingRNAi: mechanism and application
511 The origin of SiRNA Size: Size: ~20–25 base pairs in length double-stranded RNA moleculesStructure:Structure:Origin:Origin:SiRNA is a class of double-stranded RNA molecules, 20-25 base pairs in length.Small interfering RNA (siRNA)：是一种小RNA分子（~21-25核苷酸），由Dicer（RNAase Ⅲ家族中对双链RNA具有特异性的酶）加工而成。SiRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。
52Biogenesis and regulation of SiRNA 2Biogenesis and regulation of SiRNAdsRNA catalyzed by Dicer enzyme and formed 20-25bp long double strand SiRNA.SiRNA was then assembled into AGO and formed functional RISC complex.Small interfering RNA (siRNA)：是一种小RNA分子（~21-25核苷酸），由Dicer（RNAase Ⅲ家族中对双链RNA具有特异性的酶）加工而成。SiRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。
53Three ways of SiRNA-guided gene silencing3Trigger destruction of the target mRNA. (引 起靶标mRNA的降解)Inhibit translation of the target mRNA.(抑制靶标mRNA的翻译)Induce chromatin modification .(引起靶 标基因的转录沉默)
54RNAi : mechanism and application 4RNAi : mechanism and applicationWhat is RNAi ?RNAi is a process in which double-stranded RNA processing to 21–23nts siRNAs triggers the degradation of a homologous mRNA.是双链RNA（dsRNA)特异性地结合到与之序列互补 的mRNA上，导致mRNA降解，从而介导的转录水 平基因表达抑制。RNA interference(RNAi) is a process that occurs naturally within the cytoplasm inhibiting gene expression at specific sequences. Regulation of gene expression through RNAi is possible by introducing small interfering RNAs(siRNAs), which effectively silence expression of a targeted gene. RNAi activates the RNA-induced silencing complex(RISC) containing siRNA, siRNA derived from cleaved dsRNA. The siRNA guides the RISC complex to a specific sequence on the mRNA that is cleaved by RISC and, consequently, silences those genes Read more: http://www.answers.com/topic/stable-nucleic-acid-lipid-particle#ixzz3J1k33hEn
55RNAi : mechanism and application 4RNAi : mechanism and applicationThe Discovery of RNAi1998年， Andrew Fire的研究证明，在正义RNA阻断了基因表达的实验中，真正起作用的是双链RNA。这些双链RNA是体外转录正义RNA时生成的，于是提出了RNAi这个词。RNAi是在研究秀丽新小杆线虫（C. elegans）反义RNA（antisense RNA）的过程RNAi实验图片中发现的，由dsRNA介导的同源RNA降解过程。1995年，Guo等发现注射正义RNA（sense RNA）和反义RNA均能有效并特异性地抑制秀丽新小杆线虫par-1基因的表达，该结果不能使用反义RNA技术的理论做出合理解释。直到1998年，Fire等证实Guo等发现的正义RNA抑制同源基因表达的现象是由于体外转录制备的RNA中污染了微量dsRNA而引发，并将这一现象命名为RNAi。
56The question to be addressed is: Why exogenous dsRNA can inhibit the expression of genes homologous to that RNA?
57RNAi : mechanism and application 4RNAi : mechanism and applicationThe mechanism of RNAiAlthough the origin of miNRA and SiRNA is quite different.短siRNA片段（上面提到的A，B，C）解旋成单链RNA，然后单链RNA和蛋白结合形成RNA诱导的沉默复合物（RNA-Induced Silencing Complex， RISC ）。 RISC然后和与短siRNA互补的内源mRNA结合。如果这种结合具有特异性，那么内源mRNA就会被内切酶切割成不具翻译功能的无意义的RNA片段。但如果结合不具特异性，RISC复合物就黏附在mRNA上，从而阻止核糖体在内源mRNA上的移动和翻译，最终导致不能合成任何蛋白。They function through similar mechanisms.
58The heart of the RNAi mechanism 4RNAi : mechanism and applicationThe heart of the RNAi mechanism1.Dicer：RNaseIII-like multidomain ribonucleaseprocesses input dsRNA into small fragmentshelps load its small RNA products into RISC.Structural organization:---A PAZ domain, binds the endof the dsRNA---Two RNase III domains---Other non-conserved domains.2. RISCRNA induced silencing complexes : a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.
59the key component of RISC is AGO Ago conatins two unique domains：PAZ ： binds to SiRNAor miNRAPIWI ：confers sliceractivity
60RNAi : mechanism and application 4 The applification of RNAiA: Gene function research 基因功能研究For example, Applied in mammalian:Transfect exogenous siRNA into cellsExpression of siRNA in cultured cells or animal with siRNA expression vectorB: Therapeutic Applicationsa. Gene therapy 基因治疗b. Development of new drugs 新药开发
62Topic III. piRNA The definition of piRNA The feature and Biogenesis of piRNAThe Functions of piRNA
63The definition of piRNA 1 piRNA is the largest class of small non-coding RNA molecules (24-31nt) expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins.2012年前后 piRNA 研究领域开启了令人兴奋的新时代，该领域的重要成果纷纷登上Science、Nature、Cell等顶尖杂志After 2012 piRNA research opens up exciting new era, important achievements in this field have boarded Science, Nature, Cell and other top magazines.转座子能在染色体不同位点间跳跃，导致基因失活甚至引发癌症。生殖细胞系中转座子的跳跃还可能导致不孕。“对于绝大多数动物来说，无法控制转座子都会最终导致物种灭绝，”piRNA：动物基因组的大英雄
64Feature and Biogenesis of piNRA 2Feature and Biogenesis of piNRAFeatureOnly expressed in animals.DNA code for piRNA exists in clusters throughout the genome. ( piRNA clusters)The expression of PiRNAs was proved to be tissue and cell specificity.The biogenesis of piRNAs is not yet fully understood. They may derived from long single stranded precursor molecules.Biogenesis
653 The functions of piRNA in germline(生殖细胞) silencing transposable elements in the germline.(生殖细胞内的转座子沉默)Participate in the regulation of spermatogenesis and reproductive (参于精子生成和生殖调控)in non-gonadal cells (非生殖细胞)piRNAs are endogenous small noncoding RNAs ;Animals lacking piRNA functions show defects in gametogenesis and exhibit sterilitypiRNAs have also recently been shown to have roles in multigenerational epigenetic phenomena in wormsAlthough piRNAs have been previously suggested to be germline-specific, recent studies have shown that piRNAs also play crucial roles in nongonadal cells.Participate in multigenerational epigenetic phenomena In worms (参与表观遗传调控)Destruct target mRNA, silence target gene expression (破坏目标mRNA ,沉默靶基因的表达)
66Topic IV. lncRNA The definition and feature of LncRNA （2002年发现）The definition and feature of LncRNAThe origin of LncRNAThe Functions of LncRNALncRNA and diseases
67introduction to lncRNA 1introduction to lncRNADefinition：LncRNAs are non-protein coding transcripts longer than 200 nucleotides, regulates gene expression at epigenetic, transcription and post-transcription level.Feature:The expression of LncRNAs has obvious specific temporal and spatial expression during development.LncRNA broadly participates of almost all physiological and pathological processes, and is very closely associated with tumor and other diseases.The type, quantity and functions of LncRNAs are not well understood.Structure:Similar to mRNA.
68mRNA lncRNA 相 同 点 不 specific temporal and spatial expression mRNAlncRNA相同点specific temporal and spatial expressionFormation of secondary structurePost-transcriptional processing, such as the5 'cap, polyadenylation, splicingThe important role in the development and disease不code for proteinsregulatory functionHighly conserved between speciesPoorly conserved between speciesPresent in the nucleus and cytoplasmFound mainly in the nucleusTotal 20,000-24,000 mRNA3-100 times of mRNAThe expression level: Low to HighThe expression level: very low to moderate
69Interrupted protein coding genes. Chromatin reorganization. 2The Origin of LncRNAInterrupted protein coding genes.Chromatin reorganization.The reverse-displacement of Non-coding gene during replication process.Generating a series of local replicon.Insert a transposase components into a gene.蛋白编码基因的结构中断从而形成一段LncRNA染色质重组：即两个未转录的基因与另一个独立的基因串联，从而产生 含多个外显子的LncRNA非编码基因在复制过程中的反移位产生LncRNA局部的复制子串联产生LncRNA基因中插入一个转座成分而产生有功能的LncRNA
70The function of LncRNA 3 Involved in many regulation process. Protect protein-coding genes.Play an important role in embryonic development.以多种模式保护蛋白编码基因：如受到严格的功能限制以保护编码基因的ORF；或表现为基因序列的较短延伸，以保护功能域和结构such as functional limitations are strictly to protect the ORF encoding genes; or gene sequences showed shorter extension, in order to protect functional domains and structures.一些lncRNA实际上是某些调控RNA（如microRNA或piwi RNA）的前体Precursors of certain regulatory RNA(such as miRNA or piRNA)
71LncRNA and diseases4LncRNA can regulate the function of its associated protein-encoding gene, so its inappropriate expression will result in many disease, such as:Cancer (白血病、乳腺癌、肝癌、肺癌、结肠癌和前列腺癌等)cardiovascular disease （心血管疾病）neurological diseases （神经系统疾病）immune-mediated diseases（免疫介导的疾病）other relevant diseases（其它相关疾病）.Although lncRNAs are among one of the least well-understood molecules, emerging studies have revealed that lncRNAs play critical roles in a broad range of biological processes and are associated with a number of diseases, i.e. cancer, cardiovascular disease, and neurodegeneration disease. LncRNAs are becoming critically important for the understanding of life sciences, especially diseases.包括p15在内的许多因表观遗传机制而发生基因沉默的抑癌基因都可转录相应的LncRNA ，从而诱导相关疾病的发生lncRNA在多种癌症中差异表达如：白血病、乳腺癌、肝癌、结肠癌和前列腺癌等lncRNA失调还与心血管疾病、神经系统疾病和免疫介导的疾病等有关
72Summary：MiRNA、SiRNA、piRNA and LncRNA种类长度（nt）来源主要功能siRNA21-25长双链 RNA转录基因沉默miRNA21-23含发卡结构的pri-miRNApiRNA24-31长单链前体或起始 转录产物等多途径生殖细胞内转座子 的沉默lncRNA>200多种途径基因组印记和 X 染色体失活等