Main Contens Intruduction Regulation by RNAs in Bacteria Regulation by RNAs in Eukaryotes
Intruduction The discovery of regulatory RNA What is regulatory RNA ? The importance of regulatory RNA
regulatory RNAs regulatory RNAs are RNA molecules that function without being translated into proteins. What is regulatory RNA ?
Background: 人类基因组草图带给科 学家们的困惑 The discovery of miRNAs The Discovery of regulatory RNA
1. Human genome: 3 billion chemical base pairs ~20,500 genes 是果蝇的 2 倍，啤酒酵母的 4 倍 1-2 ％ code for protein 2.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. Background: 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
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. Background: 人类基因组草图带 给科学家们的困惑
8 More and more scientists believe that Non-coding RNA functions in gene-regulatory networks that underlie the complexity of higher organisms.
RNA 调控 后基因组时代的基因调控： RNA 调控
miRNA 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) Victor AmbrosGary Ruvkun
11 Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell. 2004 Jan 23;116 (2 Suppl):S93-6. Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell. 2004 Jan 23;116 (2 Suppl):S89-92 Thought to be an oddity not a general phenomenon
12 Breakthrough 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. 2000 Nov 2;408(6808):86-9.
14 The number of the identified miRNAs is growing rapidly in recent years. Over 5000 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 http://microrna.sanger.ac.uk/sequences/ and in flatfile form from ftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.
microRNAs 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.
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. The importance of regulatory RNA “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.
Regulatory RNA was first discovered in eukaryotes. Does Regulatory RNA exist in prokaryotes ?
1.Small RNAs (sRNA): regulation by base pairing. 2.Riboswitches: regulation by metabolite-mediated structure changes 3. Attenuation: Regulation by ribosome stop-mediated formation of terminators Regulation by RNAs in Bacteria
1.Small RNAs (sRNA) 21 Targets Regulation of translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively. Mechanism base pairing with the targeted sequences Regulation by base pairing with the targeted sequences on mRNAs. not requirespecific machinery Acts in trans similar to miRNA, but does not require specific machinery for action.
regulatory RNA elements direct sensors of small molecule metabolites 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 2. Riboswitches ( 核糖开关 ) Concept 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
translation initiation transcription terminationaltering the accessibility of RBS formation of terminator Regulate translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively Targets Reside 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. Mechanism Metabolite-Binding Metabolite-Binding Riboswitches Metabolite-Binding Riboswitches Metabolite-Binding
Figure a. Control of transcription termination by a riboswitch 代谢物 Figure b. Control of translation initiation by a riboswitch. 代谢物
The 2nd structures of 7 riboswitches and metabolites that they sense
target premature transcription termination switches off gene expressionamino acid biosynthetic operons 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. mechanism Requires 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.
The 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. (2) attenuation Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation The TRP operon
Fig 16-19 Transcription 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?)
30 The leader RNA and leader peptide of the trp operon
1.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. 2.The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites. 3.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.
32 Low Trp High Trp Transcription of the leader RNA. Transcription of the trp operon mRNA. RNA Pol
1. A typical negative feed-back regulation 2. Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan. 3. 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. 4. Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.
Main Contens Introduction The classification of non-coding RNA The origin, mechanism and function of regulatory RNAs in Eukaryotes
Epigenetics meiotically and mitotically heritable changes gene expression The study of meiotically and mitotically heritable changes in gene expression that are not coded for in the underlying DNA sequence. Mechanisms 1.DNA cytosine methylation 2.histone modifications 3.non-coding RNAs Concept Introduction
non-coding RNAs (ncRNAs) are RNA molecules that function without being translated into proteins. What is non-coding RNA ?
non-coding RNA are Surprisingly numerous. non-coding RNA have very different functions. non-coding RNA : non-coding RNA : Versatility in form and function Versatility in form and function
Non-coding RNA functions in gene- regulatory networks that underlie the complexity of higher organisms. Human Genome sequnce: 1-2% : coded for protein. 24% : intron sequence. What is the left 74% for? 90% is transcribed. The importantce of non- coding RNA
Housekeeping Housekeeping non-coding RNA non-coding RNA Regulatory Short Short non- coding RNA (20-500nt) Long Long non- coding RNA (500-20,000nt) SiRNA miRNA piRNA lncRNA The classification of non-coding RNA
The origin, mechanism and function of regulatory RNAs in Eukaryotes 微 Topic 1. miRNA: micro-RNA ( 微 RNA ) 小干扰 RNA Topic 2. SiRNA ： Small interfering RNA ( 小干扰 RNA ) piwi 偶联 RNA Topic 3. piRNA ： Piwi interacting RNA ( piwi 偶联 RNA ) 长非编码 RNA Topic 4. lncRNA: long non-coding RNA ( 长非编码 RNA )
1.The definition and Origin of miRNA 2.The biogenesis of miRNA 3.The action mode of miRNA 4.Multiple Roles of miRNA Topic I. miRNA
miRNAs are small ncRNAs of 22-25 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.eukaryoticnuclearbase-pairingmRNA The definition and Origin of miRNA Size: ~ ~21–23 nts Structure: short hairpin loop Origin: Derived from cellular gene 1
miRNA的生成 miRNA 基因由 Pol Ⅱ转录的 300-1000 nts 初级转录产物 pri-miRNA Pol Ⅱ Transcription Processing Functional Complex assembly The biogenesis of miRNA 2 Transcription Transcription Processing Processing Functional Complex assembly Functional Complex assembly
MiRNA binds to the target mNRA with high complementarity. MiRNA binds to the target mNRA with limited complementarity The action mode of miRNA 3
(A) miRNA in development Expression of lin-4 allows ： C. elegans to proceed to the late developmental stage lin-4 binds its target mRNAs by imperfect base pairing. Victor R. Ambros 4 Multiple Roles of miRNA
(C) miRNA controls the differentiation of the hematopoietic stem cell ( 调控造血干细胞分化 ) ( Science 2004) 3 种 miRNA 控制造血干细胞向淋巴细胞的分化过程 4 Multiple Roles of miRNA
(D) miRNA In cancer 1. miRNA are cancer genes 2. miRNA are cancer classifiers 3. miRNA are cancer drivers 4. miRNA are drugs and drug targets 4 Multiple Roles of miRNA
1.The origin of SiRNA 2.Biogenesis and regulation of SiRNA 3.Three ways of SiRNA-guided gene silencing 4.RNAi: mechanism and application Topic II. SiRNA
① The origin of SiRNA Size: ~20–25 base pairs in length Structure: double-stranded RNAdouble-stranded RNA molecules molecules double-stranded RNAmolecules Origin: Size: Structure: Origin: 1
① Biogenesis and regulation of SiRNA catalyzed by Dicer enzyme20-25bp long double strand SiRNA dsRNA catalyzed by Dicer enzyme and formed 20-25bp long double strand SiRNA.Dicer enzymeDicer enzyme assembled into AGO functional RISC complex. SiRNA was then assembled into AGO and formed functional RISC complex. 2
Three ways of SiRNA-guided gene silencing 3 Trigger 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 )
RNAi RNAi is a process in which double-stranded RNA processing to 21–23nts siRNAs triggers the degradation of a homologous mRNA. What is RNAi ? 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。 ① RNAi : mechanism and application 4
The Discovery of RNAi The Discovery of RNAi ① RNAi : mechanism and application 4
The question to be addressed is: Why exogenous dsRNA can inhibit the expression of genes homologous to that RNA?
The mechanism of RNAi ① RNAi : mechanism and application 4 origin Although the origin of miNRA and SiRNA is quite different. They function through similar mechanisms.
The heart of the RNAi mechanism RNA induced silencing complexes RNA induced silencing complexes : a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression. RNaseIII-like multidomain ribonuclease processes input dsRNA into small fragments helps load its small RNA products into RISC. Structural organization: ---A PAZ domain, binds the end of the dsRNA ---Two RNase III domains ---Other non-conserved domains. 1.Dicer ： 2. RISC ① RNAi : mechanism and application 4
the key component of RISC is AGO Ago conatins two unique domains ： PAZ ： binds to SiRNA or miNRA or miNRA PIWI ： confers slicer activity activity
Therapeutic Applications B: Therapeutic Applications The applification of RNAi ① RNAi : mechanism and application 4 A: Gene function research 基因功能研究 in mammalian: For example, Applied in mammalian: a.Transfect exogenous siRNA into cells b.Expression of siRNA in cultured cells or animal with siRNA expression vector a. Gene therapy 基因治疗 b. Development of new drugs 新药开发
① The definition of piRNA ② The feature and Biogenesis of piRNA ③ The Functions of piRNA Topic III. piRNA
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 is the largest class of small non-coding RNA molecules (24-31nt) 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. RNA piwi ① The definition of piRNA 1
Only expressed in animals. Only expressed in animals. clusters throughout the genome. DNA code for piRNA exists in clusters throughout the genome. ( piRNA clusters)genome tissue and cell specificity The expression of PiRNAs was proved to be tissue and cell specificity. Feature Biogenesis biogenesisbiogenesis may derived from long single stranded precursor molecules The biogenesis of piRNAs is not yet fully understood. They may derived from long single stranded precursor molecules.biogenesis Feature and Biogenesis of piNRA 2
The functions of piRNA transposable elements 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 ) 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 ) 3 in germline( 生殖细胞 )
1.The definition and feature of LncRNA 2.The origin of LncRNA The Functions of LncRNA 4.LncRNA and diseases Topic IV. lncRNA （ 2002 年发现）
obvious specific temporal and spatial expression The expression of LncRNAs has obvious specific temporal and spatial expression during development. participates of almost all physiological and pathological processes associated with tumor and other diseases LncRNA broadly participates of almost all physiological and pathological processes, and is very closely associated with tumor and other diseases. introduction to lncRNA Definition ： Feature: Structure : mRNA Similar to mRNA. LncRNAs are non-protein coding transcripts longer than 200 nucleotides, regulates gene expression at epigenetic, transcription and post-transcription level. 1
mRNAlncRNA相同点 ① specific temporal and spatial expression ② Formation of secondary structure ③ Post-transcriptional processing, such as the 5 'cap, polyadenylation, splicing ④ The important role in the development and disease 不同点 code for proteinsregulatory function Highly conserved between species Poorly conserved between species Present in the nucleus and cytoplasm Found mainly in the nucleus Total 20,000-24,000 mRNA3-100 times of mRNA The expression level: Low to High The expression level: very low to moderate
The Origin of LncRNA Interrupted protein coding genes. Chromatin reorganization. The reverse-displacement of Non- coding gene during replication process. Generating a series of local replicon. transposase components Insert a transposase components into a gene. 2
The function of LncRNA Precursors of certain regulatory RNA (such as miRNA or piRNA) Involved in many regulation process. Protect protein-coding genes. Play an important role in embryonic development. 3
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 ）. LncRNA and diseases LncRNA can regulate the function of its associated protein-encoding gene, so its inappropriate expression will result in many disease, such as: 4