Transcriptional Regulation of Eukaryotic
Genes (真核基因的转录调控)
1.Transcriptional initiation
2.Histone modification
3.DNA methylation
Post-Transcriptional Regulation of Eukaryotic
Genes (真核基因的转录后调控)
•RNA silencing (siRNA and miRNA)
Gene Silencing
Heterochromatin
DNA methylation and histone modifications
RNA interference (siRNA and miRNA)
Terminology
RNA Silencing
RNA interference (RNAi)
generic
terms
PTGS – post-transcriptional gene silencing
TGS– transcriptional gene silencing
Co-suppression (in plants)
Homology-dependent gene silencing
Quelling (in fungi)
siRNA/miRNA silencing
Short interfering RNA (siRNA)
 Discovery
 Biogenesis
• Sources
• Function complex assembly
 Functions
• mRNA degradation
Amplification and spread: arm race with virus
• Chromatin modification
Heterochromatin
DNA methylation
 Discovery
Inhibition by injected
anti-sense RNA
Sense RNA had
similar effects!
Jorgensen et al., Nature (1988)
Double-stranded RNA
not tested
that was how to lose
a Nobel prize!
Napoli, Lemieux, Jorgensen. Plant Cell (1990)
Double-stranded RNA is the trick!!!!!
Effect of mex-3 RNA interference on levels of endogenous mRNA
a) Negative control
b) Normal pattern of mex3 expression –embryo from a parent- uninjected
c) Embryo from a parent injected with antisense RNA
d) Embryo from a parent injected with dsRNA
Q: how did they think of using dsRNA for injection?
Fire, Xu, Montgomery, Kostas, Driver, Mello (1998), Nature 391: 806-811
 Discovery
• 长度大约为25 nt
• 正义（sense）和反义（anti-sense）转基因植株皆存在
Hamilton, Baulcombe，Science (1999)
For the discovery of RNA interference – gene
silencing by dsRNA and small RNA
David Baulcombe
RNA interference (RNAi)
 Biogenesis --- Source
• 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)
Carthew and Sontheimer, Cell (2009)
小干扰RNA(Short interfering RNA, siRNA)
是引发RNAi的充分条件，并且为Dicer的产物
P
OH
OH
P
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)
siRNAs 是Dicer作用的产物
Dicers belong to Class III of RNase III, a family of
endoribonucleases that show specificity for doublestranded 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.
The Key Players in RNA Silencing
• RNaseIII Proteins:
Dicer-1, Dicer-2, Drosha
• dsRNA-Binding Domain Proteins:
R2D2, Pasha, Loquacious
• Argonaute Proteins:
Argonaute-1, Argonaute-2, Argonaute-3,
Piwi, Aubergine
 Biogenesis --- Function complex assembly
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
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 RNAInduced Silencing Complex (RISC)
• 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
• In structure without mRNA,
guide strand nucleotides 26 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.
PIWI is an RNase H domain
The similarity was not obvious at the primary sequence level !!
• 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.
Song et al. Science 305, 1434-1437.
MID domain: the structural basis for 5’ endspecific recognition of guide RNA
• 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+.
Model for Slicer catalysis
MID
MID
MID
• 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.
Cleavage Site
Functions --- mRNA degradation
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+
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
RNA-dependent RNA polymerase
(RDR)
Q: how does off-target effect of RNAi arise?
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.
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(non-cell autonomous)的RNA沉默
与系统性(systemic)RNA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
• Host could use RNAi to become
immune to viruses
• 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
Ding and Voinnet, Cell (2007) 130, 413-422
 Functions --- RNA-Directed De novo Methylation (RdDM)
DCL3, 24 nt siRNAs, AGO4, MET1/DRM2
AGO4
Marjori A. Matzke & James A. Birchler
Nature Reviews Genetics 6, 24-35 (2005)
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。
 Functions --- RNA-Directed De novo Methylation (RdDM)
DRM1/2
Nucleic Acids Research, 2010, Vol. 38, No. 20 6883–6894
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let7/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
 Biogenesis
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
animals
Du and Zamore, Development 132, 4645-4652.
plants
• Complex loading selection
5’ terminal dependent miRNA sorting
Mallory et al., Current Opinion in Plant Biology (2008)
Functions
Functional categories of miRNA target genes
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
mRNA degradation
Carthew and Sontheimer, Cell (2009) 136, 642-655.
miRNA-dependent mRNA Degradation Through
Conventional Pathway
• 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.
Behm-Ansmant et al, Genes Dev. (2006) 20, 1885-1898
miRNA Effects Are Mediated Through
GW182 Protein
• 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)
Tritschler et al, Nat. Rev. Cell Mol. Biol. (2010) 11, 379-384
microRNA靶作用位点——在动物当中的预测和验证
•
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
Pairing to the seed is necessary
Additional pairing at nt 12-17 enhances miRNA targeting
Binding 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
The Actions Of Small RNAs In Plants
microRNA
ta-siRNAs
nat-siRNAs
ra-siRNAs
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？