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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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
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
RNA in regulation
The mechanism of RNAi and the role of miRNA in development and cancer
Topic 1: RNA interference and its mechanism
1 Double-stranded RNA inhibits expression of genes homologous to that RNA. [phenomena-现象]
2006年的诺贝尔生理学奖获得者： homologous to that RNA.
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).
Young larva (幼虫)
adult body wall at high magnification (高放大倍数的
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
adult body wall at high magnification (高放大倍数的
An plant immune system: Virus-induced gene silencing ( endogenous mRNA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]
The question to be addressed is “Why exogenous dsRNA can inhibit expression of genes homologous to that RNA?”
Exogenous dsRNA inhibit expression of genes homologous to that RNA?”
Figure 17-30 RNAi silencing
Dicer: inhibit expression of genes homologous to that RNA?”
---A PAZ domain, binds the end of the dsRNA
---Two RNase III domains
---Other non-conserved domains.
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: shows that the key component is Argonaute (AGO)
Argonaute (AGO) shows that : 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.
A model for siRNA-guided shows that mRNA cleavage by Argonaute
The multiple functions of RNAi shows that
3. MicroRNA (miRNA) & its processing shows that
MicroRNA (miRNA): shows that 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 shows that
Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails
miRNA processing shows that
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/.
Topic 2: miRNAs in animal development and other functions
1. miRNA in recent yearsC. elegans development
秀丽线虫 C. elegans
Victor R. Ambros
lin-4 recent years and let-7 miRNAs control the developmental time of C. elegans.
Expression of lin-4 recent years allows C. elegans to proceed to the late developmental stage
lin-4 recent yearsbinds its target mRNAs by imperfect base pairing.
2. miRNAs in vertebrate development: recent yearsThere are a lot unknown because the the lack of efficient methods to uncover the targets of miRNAs.
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 ( recent years控制植物表型特征)
miRNA controls the differentiation of the hematopoietic stem cell (调控造血干细胞的分化)
( Science 2004)
Some viruses encode miRNAs ( cell (有些病毒编码miRNAs)
Topic 3: miRNA in cancer
miRNAs in human: cell (
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.
Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression.
One mechanism of miRNA controlling oncogene expression reveals global changes in miRNA expression.
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 downregulate the level of miR-17-5p and miR-20a, and then analyzed the protein (B-Western) and mRNA levels (C-Northen) of E2F1.
B- downregulate the level of miR-17-5p and miR-20a, and then analyzed the protein (B-Western) and mRNA levels (C-Northen) of E2F1. 细胞淋巴瘤
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 accelerates c-myc-induced lymphomagenesis in mice.
Topic 4: siRNA application
siRNA application in mammalian accelerates c-myc-induced lymphomagenesis in mice.
Expression of hairpin RNA (shRNA) using Pol III promoters accelerates c-myc-induced lymphomagenesis in mice.
A mammalian expression vector designed to direct the intracellular synthesis of siRNAs.
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.
Key points of the chapter delivery vehicles to create
第一节： delivery vehicles to create RNA干扰