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
Chapter 8: RNA in regulation The mechanism of RNAi and the role of miRNA in development and cancer • Molecular Biology Course
CHAPTER 18 RNAi and miRNA in development and cancergenesis 一、RNA干扰及其机制 Topic 1: RNA interference and its mechanism
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
2006年的诺贝尔生理学奖获得者： Andrew Z. Fire Craig C. Mello
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)
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)
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.
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
The question to be addressed is “Why exogenous dsRNA can inhibit expression of genes homologous to that RNA?”
Exogenous dsRNA 外源双链RNA Figure 17-30 RNAi silencing
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).
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.
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
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.
RISC: the key component is Argonaute (AGO) AGO2 TRBP Dicer
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.
3. MicroRNA (miRNA) & its processing 微小RNA及其加工
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).
Structure of pri-miRNAs Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails
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
Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.
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/.
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
1. miRNA in C. elegans development 秀丽线虫 C. elegans Victor R. Ambros
Expression of lin-4 allows C. elegans to proceed to the late developmental stage
2. miRNAs in vertebrate development:There are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.
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.
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)
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)
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
miRNAs in human: There are about 500 miRNAs from human have been found and annotated. They are named as has-miRx.
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
Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression.
One mechanism of miRNA controlling oncogene expression 微小RNA调控癌基因表达的一种机制。
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)
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.
Some microRNAs are potential oncogenes 有些微小RNA可能是致癌基因。
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.
Figure 2. Overexpression of the mir-17–19b cluster accelerates c-myc-induced lymphomagenesis in mice.
CHAPTER 18 RNAi and miRNA in development and cancergenesis 四、siRNA的应用 Topic 4: siRNA application