Post-Transcriptional Regulation of Eukaryotic Genes ( 真核基因的转录后调控 )

•RNA silencing (siRNA and miRNA)

Transcriptional Regulation of Eukaryotic Genes ( 真核基因的转录调控 )1.Transcriptional initiation2.Histone modification3.DNA methylation

Gene Silencing

Heterochromatin

DNA methylation and histone modifications

RNA interference (siRNA and miRNA)

Terminology

RNA SilencingRNA SilencingRNA interference (RNAi)

PTGS – post-transcriptional gene silencingPTGS – post-transcriptional gene silencing

TGS– transcriptional gene silencingTGS– transcriptional gene silencing

Co-suppression (Co-suppression (in plantsin plants))

Homology-dependent gene silencingHomology-dependent gene silencing

Quelling (Quelling (in fungiin fungi))

siRNA/miRNA silencing siRNA/miRNA silencing

genericterms

Short interfering RNA (siRNA)Short interfering RNA (siRNA)

Discovery

Biogenesis• Sources• Function complex assembly

Functions• mRNA degradation

Amplification and spread: arm race with virus• Chromatin modification

HeterochromatinDNA methylation

Inhibition by injected anti-sense RNA

Sense RNA had similar effects!

Double-stranded RNA not tested

that was how to lose a Nobel prize!

Discovery

Napoli, Lemieux, Jorgensen. Plant Cell (1990)

Jorgensen et al., NatureNature (1988) (1988)

Double-stranded RNA is the trick!!!!!

Effect of mex-3 RNA interference on levels of endogenous mRNAa) Negative control b) Normal pattern of mex3 expression –embryo from a parent- uninjectedc) Embryo from a parent injected with antisense RNAd) Embryo from a parent injected with dsRNA

Fire, Xu, Montgomery, Kostas, Driver, Mello (1998), Nature 391: 806-811

Q: how did they think of using dsRNA for injection?

Hamilton, BaulcombeHamilton, Baulcombe ，， ScienceScience (1999) (1999)

• 长度大约为 25 nt• 正义（ sense ）和反义（ anti-sense ）转基因植株皆存在

Discovery

DavidDavid BaulcombeBaulcombe

For the discovery of RNA interference – gene silencing by dsRNA and small RNA

RNA interference (RNAi)

Carthew and Sontheimer, Cell (2009)

• Some dsRNAs have viral origin, but not all

• Genomic repetitive sequences also are source of siRNA

• Some even regulate other genes (ta-siRNA for trans-acting)

Biogenesis --- Source

PP

PP

19-nt 双链2-nt 3’ 末端突出5’- 磷酸基3’-OH: 在动物中 3’ OH 被阻碍后便失去活性植物 siRNAs 3’ 甲基化，可能保护或稳定了 siRNA分为引导链与乘客链（ Guide vs passenger strands ）具有高度保守的种子序列（ Seed region, nt 2-8)具有双链的不对称性

Tuschl T. Gene Dev 15:188 (2001)

小干扰小干扰 RNA(Short interfering RNA(Short interfering RNA, siRNA)RNA, siRNA)

是引发 RNAi 的充分条件，并且为 Dicer 的产物

OH

OH

siRNAs 是 Dicer 作用的产物

Dicers belong to Class III of RNase III, a family of endoribonucleases that show specificity for double-stranded RNA (dsRNA)----PAZ domains plus two RNase III domains

The PAZ domain• 110 aa domain found in Piwi, Ago, Zwille &

Dicer proteins• A binding pocket in PAZ accommodates the 2

nt overhang• No interactions found between the 2 nts with

the pocket, suggesting that the pocket accommodates all nucleotide combinations

Ma et al. Nature 429, 318-322 (2004)

Model for Dicer catalysis

The PAZ domain binds the 2 nt 3′ overhang of a dsRNA terminus. The RNaseIII domains form a pseudo-dimer. Each domain hydrolyzes one strand of the substrate. The binding site of the dsRBD is not specified.

A model for dsRNA processing by Dicer

The PAZ domain of Dicer, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (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.

• RNaseIII Proteins:Dicer-1, Dicer-2, Drosha

• dsRNA-Binding Domain Proteins:R2D2, Pasha, Loquacious

• Argonaute Proteins:Argonaute-1, Argonaute-2, Argonaute-3, Piwi, Aubergine

The Key Players in RNA Silencing

Loading & activation of siRNA

• siRNA 的装载需要一个双链 RNA 结合蛋白R2D2 。 R2D2 包含两个一前一后的双链 RNA 结合结构域

• Dicer-2 与 R2D2 形成了一个异源二聚体• R2D2 将与 siRNA 热稳定性更高的一端结合（ 3’ 端）• siRNA 的不对称性（ siRNA asymmetry ） : 引导链

的 5’ 端稳定性较差

Biogenesis --- Function complex assembly

Asymmetrical loading & activation of siRNAs

R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RISC-loading complex (RLC). As siRNA unwinding proceeds, the heterodimer is exchanged for Argonaute-2, the core component of the RISC. Dcr-2 and R2D2 are envisioned to recruit Ago2 directly to the double-stranded siRNA. Ago2 exchanges first with Dcr-2, with whom it makes a protein-protein contact, then with R2D2. Finally, Ago2 cleaves the passenger strand (blue), thereby liberating the guide (red) from the siRNA duplex and producing active RISC.

Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)

Carthew and Sontheimer, Cell (2009) 136, 642-655.

• In structure without mRNA, guide strand nucleotides 2-6 have bases exposed and available for base-pairing

• PIWI domain adopts RNase H fold and in some Ago proteins can cleave the ‘passenger strand’ : I.e. the mRNA

Carthew and Sontheimer, Cell (2009) 136, 642-655.

• One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute

• Lobes formed by PAZ domain and PIWI and Mid domains

The similarity was not obvious at the primary sequence level !!

Song et al. Science 305, 1434-1437.

PIWI is an RNase H domain

• The tertiary structure of the piwi domain core belongs to the RNase H family of enzymes. One characteristic of the structure is a five-stranded mixed β sheet surrounded by helices.

• The RNA/RNA* duplex is positioned in a basic channel spanning the MID-PIWI interface

• The 5’ P is inserted into a conserved basic pocket located primarily within domain MID and the C terminus of domain PIWI.

• The 5’ P is anchored by a network of hydrogen bonds involving the side chains of a number of amino acids and a divalent cation, Mg2+.

MID domain: the structural basis for 5’ end-specific recognition of guide RNA

• The siRNA guide strand is bound at the 5′ end by the MID/PIWI domains and at the 3′end by the PAZ domain.

• mRNA targets are initially bound by the seed region of the siRNA and pairing is extended to the 3′end.

• Slicer cleavage is measured from the 5′end of the siRNA.

Model for Slicer catalysis

MIDMID MIDMID MIDMID

Cleavage Site

In vitro Demonstration of Slicer Activity

• Human Ago2 mixed with 2 siRNAs and a 500-nt RNA target

• Products of expected size were produced, dependent on siRNA, target RNA, and Mg2+

Functions --- mRNA degradation

In Some Organisms, siRNA Signal Is Amplified and Spread

• New siRNAs appear against other regions of targeted mRNAs

• Signal can even spread to other cells in plants and nemotodes

• Amplification does not seem to be present in animals

RNA 依赖的 RNA 聚合酶（ RNA-dependent RNA polymerase ， RDRP)

• 最早克隆自被类病毒侵染的番茄中 (1998, Plant Cell)• 与 RNA 病毒编码的 RdRP 基本没有同源性• 在果蝇和哺乳动物基因组中不存在• 是产生次级小干扰 RNA 的放大效应的主要因子

a, RNAs are normally not silenced because the RDR proteins do not have access to the template RNA sequence. Cap-binding protein (CBP) and poly-adenosine-binding protein (PABP) may be involved in this restriction of RDR access. However in b, the RDR protein is allowed access because the RNA lacks a 5' cap or 3' poly-adenosine tail, and dsRNA is produced which enters the siRNA pathway. b, The amplification process would result from the ability of a single aberrant RNA to generate many molecules of siRNA. c shows the outcome if a small quantity of primary siRNA is present from either a virus, a transposon or from a cellular RNA through the process shown in b. The antisense strand of this siRNA may anneal by base pairing to a target RNA and serve as a primer for the RDR. The resulting dsRNA would then be cleaved by Dicer and, as in b, there would be amplification because many secondary siRNAs would be produced from each molecule of primary siRNA.

RNA-dependent RNA polymerase (RDR)

Q: how does off-target effect of RNAi arise?

RNAi 的生物学意义

参与基因表达调控

保护基因组免受外源核酸侵入

维持基因组稳定

Short and long range cell to cell movement of RNAi

非细胞自主非细胞自主 (non-cell(non-cell autonomous)autonomous) 的的 RNARNA 沉默沉默与系统性与系统性 (systemic)RNA(systemic)RNA 沉默沉默

RNAi 作用可以向其它细胞传递，因此受到其他细胞或组织传来的小 RNA 的抑制成为非细胞自主的RNA 沉默，可以分为两类：

细胞与细胞间的传递：包括近程与远程两种

系统性传递：通过传输器官（如韧皮部）在不同组织间广泛传播。

The mobile signal is likely to be siRNA or dsRNA

An Arms Race Between Virus and Host

Ding and Voinnet, Cell (2007) 130, 413-422

• Viruses could also use RNAi to compromise host defense

• Why not mutate so that a viral sequence corresponds to host defense-related genes?

• Viruses have most likely done this, and there is likely a continual arms race

• Even amplification and spread of an siRNA signal could be hijacked by viruses to ‘prime’ new cells for easy infection

VSR: Viral Suppressors of RNA silencing

• Host could use RNAi to become immune to viruses

Functions --- RNA-Directed De novo Methylation (RdDM)

Marjori A. Matzke & James A. BirchlerNature Reviews Genetics 6, 24-35 (2005)    

DCL3, 24 nt siRNAs, AGO4, MET1/DRM2

AGO4AGO4

RdDM 主要通过 dsRNA 介导相同 DNA 序列发生重新甲基化 (de novo methylation) 而实现转录水平的基因沉默。

• 引起相同序列 DNA 甲基化的物质是 dsRNA

• RdDM 的生物学意义 : 阻抑不必要基因（ repetitive sequences) 和有害基因

( 尤其是 transposons) 的表达，对维护基因组的稳定至关重要。

Nucleic Acids Research, 2010, Vol. 38, No. 20 6883–6894

DRM1/2

Functions --- RNA-Directed De novo Methylation (RdDM)

Micro RNA (miRNA)Micro RNA (miRNA)

Discovery

Biogenesis• Biogenesis• Complex loading selection

Compare with siRNA

Functions• mRNA degradation• Ribosome drop-off• Initiation block

Technical application• Artificial miRNA

Discovery

• 1993 年 ,Lee RC等在线虫 (C.elegans)中意外地发现了一种定时调控胚胎后期发育的miRNA-lin4,它是一种非编码 RNA,长度为22 nt。

• 2000 年 ,miRNA-let7的发现掀起了寻找miRNA的热潮。

在线虫（ C. elegans）当中 ,通过功能缺失突变体的筛选，找到了let-7/lin-41基因

不同物种中的 let-7 基因具有序列保守性 ,且均可与 lin-41 基因的 3’UTR 区域互补

在 lin-41 基因的 3’UTR 区域发现了 let-7 的互补区

Transcription of miRNA genes by RNA Pol II

• Transcribed by pol II to pri-miRNA (primary precursor)– Pri-miRNA contains the 7-methylguanosine cap and a poly(A) tail

– Pol II is physically associated with miRNA gene promoters

– miRNA gene transcription is sensitive to -amanitin

• Pol II dependent transcription enables temporal and spatial regulation of miRNA production.

Biogenesis

Du and Zamore, Development 132, 4645-4652.

animals plants

Biogenesis

• Complex loading selection

Mallory et al., Current Opinion in Plant Biology (2008)

5’ terminal dependent miRNA sorting

Functional categories of miRNA target genes

Functions

microRNA 的作用机理

• Reduction of mRNA stability– Plant miRNAs guide cleavage of target mRNAs (however only

a few miRNA targets have been examined at the protein level)– Animal miRNAs also reduce stability of target mRNAs

• Inhibition of mRNA translation– lin-4 mediated regulation of lin-14; let-7 mediated regulation of

lin-41– Other animal miRNAs cause reduced target protein levels

without affecting target mRNA levels in cell culture– At least three examples of plant miRNAs affecting target

protein but not mRNA levels

• mRNA degradation

Carthew and Sontheimer, Cell (2009) 136, 642-655.

mRNA degradation

miRNA-dependent mRNA Degradation Through Conventional Pathway

Behm-Ansmant et al, Genes Dev. (2006) 20, 1885-1898

• Izaurralde and co-workers (2006)

• mRNA decay followed in Drosophila cells

• Actinomycin D used to block transcription, reporter mRNA level then followed by Northern blot

• Decay of mRNA dependent on GW182, Ago, and deadenylation (NOT1/CAF1) and decapping (DCP) machineries

GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation.

miRNA Effects Are Mediated Through GW182 Protein

Tritschler et al, Nat. Rev. Cell Mol. Biol. (2010) 11, 379-384

• GW182 shown to be required for miRNA effects

• Also sufficient: tethering GW182 without Ago gave silencing

• GW182 shown to bind to polyA-binding protein (PABP or PABPC1 here)

• 动物 miRNAs 只与它们的靶位点保持很低的互补性：仅为 miRNAs 中 2-7 个核苷酸 ( 成为种子区 seed sequence) ，且决定了 miRNA 的功能。– Within miRNA target sites of invertebrate miRNAs, residues that

pair with nucleotides 2-7 of the miRNAs are conserved in orthologous mRNAs of other species.

– Nucleotide 2-7 of the miRNA are the most conserved among homologous metazoan miRNAs.

– Experimental evidence also indicates that nucleotides 2-7 in siRNAs are more important than others in guiding cleavage

microRNA 靶作用位点——在动物当中的预测和验证

Pairing to the seed is necessaryAdditional pairing at nt 12-17 enhances miRNA targetingBinding site location preference ： Local AU rich region

• 植物 miRNAs 和靶 mRNA 具很高序列互补性，因此可以利用类似于 siRNA 介导的剪切机制去剪切靶 mRNAs

• 如何研究 miRNA 的功能？miRNA 基因敲除突变体miRNAs 基因过表达突变体在内源启动子作用下表达 miRNA-resistant targets 约半数预测的保守 miRNA 的靶位点都存在于转录因子的 mRNAs 中 ( 转录因子只占基因的 6%)

miRNA 的生物学功能

• Plant miRNAs

– Many act in cell differentiation and developmental patterning by targeting transcription factor mRNAs

– Other miRNAs target non-transcription factor mRNAs and may play a role in physiological processes or stress responses

– Essential functions of miRNAs illustrated by the embryo lethal phenotype of dcl1 null mutants

• Animal miRNAs

– Developmental patterning

• ES cells lacking Dicer are viable but cannot differentiate in vitro and in vivo.

• Dicer knockout zebrafish lacking both maternal and zygotic Dicer have intact patterning in the first 24 h but fail to continue with morphogenesis

– Physiological functions

– Cancer

microRNA ta-siRNAs nat-siRNAs ra-siRNAs

The Actions Of Small RNAs In Plants

Vazquez 2006

Key Points

• Small RNAs, in the forms of siRNAs and miRNAs, play large roles in the regulation of gene expression in eukaryotes. They are important in normal cell metabolism, development, and defense against invaders.

• siRNAs are produced from longer segments of dsRNA by Dicer, assembed into RISC, and targeted to mRNAs with perfect complementarity, giving silencing by cleavage and degradation of the RNA or by formation of heterochromatin.

• The pathway for miRNA has many steps in common with that for siRNA. However, miRNAs are processed from hairpin structures by Drosha and then by Dicer, and they most often have imperfect complementarity with their targets, giving effects on translation rather than Ago-mediated cleavage of the mRNA.

思考题

1 ，如何判断 miRNA 在 mRNA 翻译的哪个阶段（起始、延伸或终止）行使翻译抑制功能？

2 ，在 Fire 等的 Nature 文章中，他们是怎样想到用 dsRNA 的？

3 ， siRNA 的 off-target effect 是如何发生的？

4 ， 转 sense CHS gene 为什么会产生红白相间的牵牛花？