Welcome each of you to my molecular biology class
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Welcome Each of You to My Molecular Biology Class. Molecular Biology of the Gene, 5/E --- Watson et al. (2004). Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods. 2005-5-10.

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Welcome Each of You to My Molecular Biology Class

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Welcome each of you to my molecular biology class

Welcome Each of You to My Molecular Biology Class


Welcome each of you to my molecular biology class

Molecular Biology of the Gene, 5/E--- Watson et al. (2004)

Part I: Chemistry and Genetics

Part II: Maintenance of the Genome

Part III: Expression of the Genome

Part IV: Regulation

Part V: Methods

2005-5-10


Welcome each of you to my molecular biology class

Chapter 16 Regulation principles and How genes are regulated in bacteria

Chapter 17 Basic mechanism of gene expression in eukaryotes

Chapter 18 The mechanism of RNAi and the role of miRNA in development and cancer


Welcome each of you to my molecular biology class

Chapter 8:

RNA in regulation

The mechanism of RNAi and the role of miRNA in development and cancer

  • Molecular Biology Course


Welcome each of you to my molecular biology class

CHAPTER 18 RNAi and miRNA in development and cancergenesis

一、RNA干扰及其机制

Topic 1: RNA interference and its mechanism


Welcome each of you to my molecular biology class

1 Double-stranded RNA inhibits expression of genes homologous to that RNA. [phenomena-现象]

双链RNA抑制含其同源序列基因的表达


Welcome each of you to my molecular biology class

2006年的诺贝尔生理学奖获得者:

Andrew Z. Fire

Craig C. Mello


Welcome each of you to my molecular biology class

Fig 2. Analysis of RNA-interference effects in individual cells. Fluorescence micrographs show progeny of injected animals from GFP-reporter strain PD4251 (a C. elegans strain expressing GFP fluorescence protein) (使用外源导入的报告基因).

ds-gfp RNA

Control dsRNA

Young larva (幼虫)

Adult (成虫)

adult body wall at high magnification (高放大倍数的

成虫体壁)


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Fig 3. Effects of mex-3 RNA interference on levels of the endogenous mRNA (in situ hybridization in embryos) (胚胎的原位杂交).

+ hybridization (endogenous mex-3 RNA)

No hybridization and staining

+ds mex-3 RNA

+hybridization

+antisense

+hybridization

adult body wall at high magnification (高放大倍数的

成虫体壁)


Welcome each of you to my molecular biology class

An plant immune system: Virus-induced gene silencing (植物病毒引起的基因沉默).

Most plant viruses have single-stranded RNA genomes, which are released from the protein coat of their virus particles as they enter a cell.Their genomic RNA is then replicated by the virus encoded RNA-dependent RNA polymerase to produce sense and antisense RNA, which can hybridize to form dsRNA and trigger an RNAi response against their own sequences.


Welcome each of you to my molecular biology class

2. Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way. [Mechanism-机制]

从双链RNA产生的小干扰RNA可以指导用不同机制关闭基因的细胞机器


Welcome each of you to my molecular biology class

The question to be addressed is “Why exogenous dsRNA can inhibit expression of genes homologous to that RNA?”


Welcome each of you to my molecular biology class

Exogenous dsRNA

外源双链RNA

Figure 17-30 RNAi silencing


Welcome each of you to my molecular biology class

  • The targets of the RNAi-directed gene silencing

  • Degradation of the target mRNA (引起靶标mRNA的降解),

  • Inhibition of translation of the target mRNA (抑制靶标mRNA的翻译),

  • Silencing the gene transcription from the target promoter (引起靶标启动子的转录沉默).


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  • The heart of the RNAi mechanism

  • Dicer: an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments called short interfering RNAs (siRNAs) or microRNAs (miRNA). Dicer then helps load its small RNA products into RISC.

  • RISC(RNA induced silencing complexes) (RNA诱导的沉默复合体): a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.


Welcome each of you to my molecular biology class

Dicer:

Structural organization:

---A PAZ domain, binds the end of the dsRNA

---Two RNase III domains

---Other non-conserved domains.

贾第鞭毛虫


Welcome each of you to my molecular biology class

The crystal structure of the Giardia intact Dicer enzyme shows that the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic RNase III domains by a flat, positively charged surface.

The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.


Welcome each of you to my molecular biology class

RISC: the key component is Argonaute (AGO)

AGO2

TRBP

Dicer


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Argonaute (AGO): A large protein family that constitutes key components of RISCs.

---AGO proteins are characterized by two unique domains, PAZ and PIWI, whose functions are not fully understood. Current evidence suggests that the PAZ

domain binds the 3’-end two-nucleotide overhangs of the siRNA duplex, whereas the PIWI domain of some AGO proteins confers slicer activity. PAZ and PIWI

domains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or translational repression.

---Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the

mRNA target, whereas AGO1 and AGO3 do not.


Welcome each of you to my molecular biology class

A model for siRNA-guidedmRNA cleavage by Argonaute


Welcome each of you to my molecular biology class

The multiple functions of RNAi


Welcome each of you to my molecular biology class

3. MicroRNA (miRNA) & its processing

微小RNA及其加工


Welcome each of you to my molecular biology class

MicroRNA (miRNA):A type of non-coding small RNA (~21–23 nucleotides) produced by Dicer from a stem-loop structured RNA precursor (~70-90 nts ong) (结构和来源). miRNAs are widely expressed in animal and plant cells as RNA–protein complexes, termed miRISCs, and have been implicated in the control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA (生物学功能和机制).


Welcome each of you to my molecular biology class

Structure of pri-miRNAs

Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails


Welcome each of you to my molecular biology class

miRNA processing

Pri-miRNA

(miRNA初级转录产物)

Drosha

(1)

pre-miRNA

(miRNA前体)

Dicer

(2)

miRNA

Exportin 5 (Exp5) transports pre-miRNA to the cytoplasm


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Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.


Welcome each of you to my molecular biology class

The number of the identified miRNAs is growing rapidly in recent years. Over 4000 miRNAs have been found until the October of this year (The miRBase Sequence Database). Release 9.0 (Oct, 2006) of the database contains 4361 entries representing hairpin precursor miRNAs, expressing 4167 mature miRNA products, in 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/.


Welcome each of you to my molecular biology class

CHAPTER 18 RNAi and miRNA in development and cancergenesis

二、MicroRNA在发育中的调控作用,及其他作用

Topic 2: miRNAs in animal development and other functions


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1. miRNA in C. elegans development

秀丽线虫 C. elegans

Victor R. Ambros


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lin-4 and let-7 miRNAs control the developmental time of C. elegans.


Welcome each of you to my molecular biology class

Expression of lin-4 allows C. elegans to proceed to the late developmental stage


Welcome each of you to my molecular biology class

lin-4binds its target mRNAs by imperfect base pairing.


Welcome each of you to my molecular biology class

2. miRNAs in vertebrate development:There are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.


Welcome each of you to my molecular biology class

Learning the miRNA function from its expression pattern

果蝇

小鼠

青鳉

斑马鱼

Figure 2. Expression of miR-124a and miR-1 in Zebrafish, Medaka, Mouse, and Fly.

miR-124a is restrictedly expressed in the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly. The expression of miR-1 is restricted to the muscles and the heart in the mouse.


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miRNA controls some plant phenotype (控制植物表型特征)

Jaw-miRNA 控制拟南芥叶形变化

(Nature, 2003)


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miRNA controls the differentiation of the hematopoietic stem cell (调控造血干细胞的分化)

3种miRNA控制造血干细胞向淋巴细胞的分化过程

( Science 2004)


Welcome each of you to my molecular biology class

Some viruses encode miRNAs (有些病毒编码miRNAs)


Welcome each of you to my molecular biology class

CHAPTER 18 RNAi and miRNA in development and cancergenesis

三、微小RNA在癌症发生中的作用

Topic 3: miRNA in cancer


Welcome each of you to my molecular biology class

miRNAs in human:

There are about 500 miRNAs from human have been found and annotated. They are named as has-miRx.


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miRNA expression pattern changes during oncogenesis, and is unique for each cancer.

微小RNA在癌症发生中表达谱的变化


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Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression.


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One mechanism of miRNA controlling oncogene expression

微小RNA调控癌基因表达的一种机制。


Welcome each of you to my molecular biology class

  • c-Myc is a helix–loop–helix leucine zipper transcription factor that regulates an estimated 10–15% of genes in the human and Drosophila genomes.

  • c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. (Figure 1)

  • E2F1 is the transcription factor, which is a target of c-Myc that promotes cell cycle progression.

  • Expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. (Figure 1)


Welcome each of you to my molecular biology class

Used 2’-O-methyl Antisense oligonucleotides to downregulate the level of miR-17-5p and miR-20a, and then analyzed the protein (B-Western) and mRNA levels (C-Northen) of E2F1.


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Some microRNAs are potential oncogenes

有些微小RNA可能是致癌基因。


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B-细胞淋巴瘤


Welcome each of you to my molecular biology class

Figure 1. The mir-17–92 cluster shows increased expression in B-cell lymphoma samples and cell lines.The level of mir-17–92 pri-miRNA was determined by real-time quantitative RT-PCR in 46 lymphomas and 47 colorectal carcinomas, and compared to levels found in corresponding normal tissues from five individuals.


Welcome each of you to my molecular biology class

Figure 2. Overexpression of the mir-17–19b cluster accelerates c-myc-induced lymphomagenesis in mice.


Welcome each of you to my molecular biology class

CHAPTER 18 RNAi and miRNA in development and cancergenesis

四、siRNA的应用

Topic 4: siRNA application


Welcome each of you to my molecular biology class

siRNA application in mammalian

  • Transfect exogenous siRNA into cells (transient expression)

  • Chemical synthesis: expensive

  • In vitro transcription of pre-miRNA with T7 promoter.

  • In vitro transcription of long dsRNA by that are then cleaved by E. coli RNase III or RNase III-like DICER.

  • Expression of siRNA in cultured cells or in animal models

  • siRNA produced with pol III promoter from the transfected DNA plasmids.


Welcome each of you to my molecular biology class

Expression of hairpin RNA (shRNA) using Pol III promoters

  • Transcription from RNAP III promoters of U6 and H1 are well characterized. RNAP III transcription uses a well-defined termination signal (TTTTT) and the products have no extra sequence.

  • 2. Transcription from these promoters is very efficient in various tissues.


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A mammalian expression vector designed to direct the intracellular synthesis of siRNAs.


Welcome each of you to my molecular biology class

  • Create induced phenotypes that can be observed over long time spans

  • Create a stably engineered cells can be assayed either in vitro or in vivo, perhaps testing the angiogenic (血管生成)or metastatic (转移)potentials of tumor cells in xenograft models (异种移植模型)。

  • Create hypomorphic alleles (亚等位基因) rapidly in transgenic mice.


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4. Combine shRNAs with existing high-efficiency gene delivery vehicles to create bona fide RNAi-based therapeutics. For example, ultimately, to silence a disease-causing mutant allele specifically.


Research applications of rnai a new strategy of reverse genetics a novel way of gene knock out

Research Applications of RNAi: A new strategy of reverse genetics & a novel way of gene knock-out

  • It can be used in reverse genetics (反向遗传学) to identify the cellular or biological function of a gene.

  • It can be combined with genomics to perform large-scale genetic screens aimed at gene discovery.


Therapeutic applications of rnai a new strategy to invitation of new drugs and gene therapy

Therapeutic Applications of RNAi:A new strategy to invitation of new drugs and gene therapy

  • siRNAs can be used to counter viral infection by specifically destroying the mRNAs of the pathogenic viruses, such as HIV and HBV.

  • siRNAs can be applied to counter cancers by specifically down-regulate the expression of genes related to oncogenesis.


Welcome each of you to my molecular biology class

Key points of the chapter

  • RNA interference: siRNA, miRNA and RNAi mechanism.

  • miRNAs functions: animal development etc.

  • miRNA in cancer: biomarker, mechanisms

  • siRNA application: methods to produce siRNA, the application of siRNA in research and therapeitics.


Welcome each of you to my molecular biology class

第一节:RNA干扰

重点:RNA干扰,小干扰RNA(siRNA)与微小RNA(miRNA)的加工和RNA干扰机制。

第二节MicroRNA在发育中的调控作用,及其他作用

重点:微小RNA在秀丽线虫发育中的作用。

第三节 微小RNA在癌症发生中的作用

内容:人类微小RNA的介绍;微小RNA表达谱在不同癌症病变中的变化;微小RNA调控癌基因表达的机制。

第四节 siRNA的应用

重点:siRNA的产生方法及其在科研和治疗上的应用。


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