Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by ...
RNA Silencing Poster
Transcript of RNA Silencing Poster
DOI: 10.1126/science.309.5740.1518a , 1518a (2005); 309Science
Poster: RNA Silencing
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RNAse IIIdsRNA-specific nucleases
ArgonauteCore proteinSome with catalytic activity
Neurospora crassaTetrahymenaVertebrateMouse
Fruit flyDrosophila melanogaster
NematodeCaenorhabditis elegans
Fission yeastSchizosaccharomyces pombe
PlantPetunia, Tobacco, Arabidopsis thaliana
HelicasesDestabilize dsRNAs
RdRpRNA-dependent RNA polymerases
dsRNA binding proteinsRNAse III associated
Others
Ago1RDE-1 (RNAi)
ALG-1 and ALG-2 (miRNA)
PPW-2 (transposon silencing)
PPW-1 (germline RNAi)
Ago1Ago2 (endonuclease)
Ago3Ago4
DCR-1 (RNAi, miRNA)Drosha (miRNA)
DRH-1/DRH-2 (RNAi)
SMG-2 (RNAi)
MUT-14 (transposon silencing,
germline RNAi)
RRF-1 (somatic RNAi)
QDE-2 (Quelling)
Aubergine (Stellate silencing,
heterochromatin silencing)
piwi (heterochromatin, cosuppression)
Ago1 (miRNA)
Ago2 (siRNA)
EGO-1 (germline RNAi)
RRF-3 (RNAi silencer)
RDE-4 (RNAi)
R2D2 (siRNA)
Pasha (pre-miRNA)
Loquacious (miRNA)
ERi-1 (RNAi)
RDE-3 (RNAi)
SID-1 (RNA transporter, RNAi)
MUT-7 (cosuppression, germline RNAi)
MUT-8 (transposon silencing, RNAi)
VIG-1 (miRNA)
TSN-1 (miRNA)
QDE-1 (Quelling)
QDE-3 (Quelling)
AGO1 (miRNA, PTGS*)
SGS2/SDE1/RDR6 (virus, PTGS*)
SDE3 (VIGS, PTGS*) Hrr1
Spindle-E (Stellate silencing,
heterochromatin)
Armitage (RISC formation)
Dcr1
Rdp1
Cid12
Dicer (RNAi and miRNA,
essential)
Drosha (miRNA)
Tudor-SN (RNAi and
miRNA) VIG (RNAi and miRNA)
FXR (RNAi and miRNA)
Tudor-SN (RNAi and miRNA)
AGO4 (heterochromatin)
DCL1 (miRNA)
DCL2 (virus related)
DCL3 (heterochromatin)
Hyl1 (miRNA)
DRB4
RDR1 (virus PTGS*)
*PTGS, posttranscriptional gene silencing
RDR2 (chromatin silencing)
DGCR8TRBP
Dicer-2 (RNAi)
Dicer-1 (miRNA)
Drosha (miRNA)
Gemin3 (miRNP)
(miRNA)
HEN1 (miRNA, RNA methylase)
Twi1
SGS3 (PTGS*)
SDE4/PoI IV subunits (RNAi) Exportin-5 (miRNA
transporter)
Gemin4 (miRNA)
Dicer (DCL1)
HASTY (miRNA transporter)
Pdd1
Twi1
Transcription of the genome
dsRNA
Exportation of scnRNAs into old macronucleus
Degradation ofscnRNAs with homology
Migration of remaining scnRNAs intonew macronucleus
Micronucleus
scnRNAs
Twi1
Twi1 Twi1
MeMe
Histone H3–lysine 9 methylation
The ciliated protozoan Tetrahymena
contains two nuclei. During somatic
growth, the germline micronucleus is
transcriptionally silent. About 15% of
the micronuclear genome consists of
germline-specific sequences (yellow) that are
eliminated when the somatic macronucleus is
formed. All gene expression occurs in the macronucleus
from the macronuclear-destined sequences (blue).
To accomplish this elimination, Dcl1 cleaves dsRNA molecules
formed by the bidirectional transcription of the micronuclear
genome into small “scan” RNAs (scnRNAs). The scnRNAs form
complexes with the Argonaute protein Twi1 and are transferred
into the old macronucleus, where scnRNAs with homology to
the macronuclear genome are degraded.
The remaining complexes, derived from micronuclear-specific
sequences, are exported to the new macronucleus where they target
methylation to their homologous sequences, which, in turn, recruits
Pdd1. The germline-specific sequences are then removed from the
macronucleus, leaving only macronuclear-specific sequences (blue).
Dicer
Pdd1
Ago1
Chp1
Tas3
Tas3
In plants, TGS often involves
DNA methylation. siRNAs are
implicated in the de novo
methylation of gene
sequences—including
non-CG methylation and CNG
methylation by different
complexes.
In fruit flies, RNAi-associated
genes such as piwi and
aubergine (two Argonaute
proteins) and spindle-E (an
RNA helicase) are involved in
TGS. Mutation in these genes
results in a loss of hetero-
chromatin. Piwi also has a
central role in cosuppression
in flies.
In fission yeast, the RNA-induced
transcriptional silencing (RITS) complex
contains centromere-specific siRNAs, an
Argonaute protein (Ago1), a chromodo-
main protein (Chp1), and Tas3.
RITS associates with chromatin
through siRNA-nascent transcript
base pairing, as well as binding to
H3–lysine 9 methylated nucleosomes. Clr4
RITScomplex
Once bound to chromatin, RITS
recruits another protein complex
that contains RdRP and two other
RNA-modifying enzymes (Cid12,
Hrr1). Together these proteins
initiate and maintain the silent
heterochromatin packaging on
centromeric regions of the
chromosomes.
Chp1
Tas3Me
Me
Chp1
Hrr1Cid12
RdRp
ViraldsRNA
Viral RNA
ViralsiRNA
Potyvirus
Ago
Ago
Blocks RISC complex formation
Blocks RISCactivityHc-Pro
Plant and likely animal cells direct siRNAs
against invading viruses to prevent or slow viral
replication. In the ongoing arms race between
host and pathogen, many plant and some
animal viruses in turn have evolved proteins,
suppressors of gene silencing, which interfere
with different steps of the RNAi machinery.
Ago
Hc-Pro, the RNAi
suppressor of the
Potyvirus family, which
infects plants, blocks
RISC activity rather than
affecting RISC formation.
The best-characterized silencing suppressor is
the p19 protein (above) of the Tombusvirus
family, which infects plants. Homodimerized
p19 sequesters siRNA, preventing the
formation of active RISC and consequent
silencing.
Tombus-virus
p19
p19
12
Dicer
MicroRNAs are encoded in the genome and produced by the maturation of
a hairpin-shaped RNA transcript. These RNAs are key regulators of many
biological processes such as development (see example in Zebrafish at
top right), cell proliferation, apoptosis, morphogenesis, oncogenesis (see
example in mouse at bottom right), and hematopoiesis. In animals, they
generally work by blocking protein synthesis or destabilizing mRNA. In
contrast, miRNAs from plants and some from animals predominantly
induce mRNA degradation and are important for plant siRNA formation.
They also act as defense mechanisms by targeting RNA from viruses,
leading to silencing of viral protein expression. MicroRNAs produced
by viruses can also affect expression of host genes.
Ventral view of mir-140 expression in Zebrafish embryos.
Overexpression of mir-17-19b clusters accelerates lymphomas formation in mice.
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Bacteriaexpressing
dsRNA
F1generation
F2generation
Virus
ViraldsRNA
HostsiRNAs
SDE3
SID-1
C. elegans can take up dsRNAs by ingestion of
bacteria expressing dsRNA molecules (which
requires a cellular factor called SID-1); by
being soaked in a solution containing dsRNA;
or by being injected with dsRNA into the body
cavity. Gene silencing spreads to most cells of
the animal, with the exception of neurons.
Moreover, RNA silencing can be passed on for
several generations. The physiological role of
spreading in C. elegans is unknown.
RNA silencing in plants
serves as an antiviral
mechanism. Viral infection
produces double-stranded
RNA (dsRNA) intermediates,
which are processed by the
host RNAi machinery into
small interfering RNAs
(siRNAs). Spreading of the
silencing signal throughout
the plant ensures that, when
the plant is later exposed to
the same virus, it will be
resistant to further infection.
In plants there are two types
of spreading: short-range
transmission of siRNAs from
cell to cell (involving SDE3)
and long-range transmission
of an unknown substance
through phloem.
TriggerSu(Ste) on Y chromosome
Tandem repeats
TargetStellate on X chromosome
Tandem repeats
dsRNA
rasiRNAs25–27 nt
Aubergine?
TGS?
mRNAcleavage
?
RISCcomplex
In the testis of male fruit flies, overexpression of the repetitive Stellate genes on the X chromosome is prevented by the
Suppressor of Stellate [Su(Ste)] locus on the Y chromosome. Overlapping sense and antisense RNAs are generated from
the Su(Ste) locus, which form dsRNAs. Dicer processes these dsRNAs into short repeat-associated siRNAs (rasiRNAs), with
the possible assistance of two RNA helicases Spindle-E and Armitage, and the dsRNA binding protein Loquacious. The
rasiRNAs are probably incorporated into a RISC-like complex with Aubergine, an Argonaute homolog, which silences the
Stellate genes.
AAAAA
A
Dicer
RNAi Suppressors VIRUSES STRIKE BACKSome viruses have evolved endogenous proteins that interfere with the gene silencing machinery, as a countermeasure to attenuate host antiviral defenses.
MicroRNAs MICROCONTROLLERS OF MULTIPLE PATHWAYSThese noncoding genes, found in nearly every eukaryotic organism, are often highly conserved through evolution and are involved in regulation of a diverse range of biological pathways.
DNA Elimination RNA-DIRECTED GENOME SHREDDINGIn this unusual process that may function as a germline defense mechanism,small “scan” RNAs generated by an RNAi-like process direct the elimination of DNA segments.
Stellate Silencing RNAi DOIN’ IT NATURALLYSilencing of the repetitive Stellate genes in Drosophila melanogaster, which isnecessary for male fertility, was the first example of endogenous dsRNA-mediated gene repression.
Small RNAs from both inside and outside the cell are processed by theRNA interference (RNAi) machinery to inhibit genes and proteins by cleavingmessenger RNAs, blocking protein synthesis, or inhibiting transcription.
CleavageDicer cleaves long
dsRNA into 21 - to 27- nucleotideintervals processively.
RISC loading complexOne strand of the siRNA duplex is loaded
into the RISC complex. The less complementary, and thus less stable, 5’ end will unwind more
easily. That strand will be incorporated into RISC. The other strand is eliminated.
RISC complexThe RNA-induced silencing complex (RISC) is the central element of all RNA
silencing pathways. It contains at least one Argonaute protein and a small noncoding RNA. This complex carries out one of three silencing operations, as dictated by
its specific RNA: mRNA cleavage, protein synthesis block, or transcriptional gene silencing (TGS).
siRNA fully complementary
to mRNAsiRNA partially
mismatched with mRNA
TGS
Protein synthesis block
miRNAs cooperatively bind to 3’ untranslated region (3’UTR) elements with imperfect complementarity and
prevent translation or destabilize the mRNAs.
RISC-induced sequence-specific cleavage of the target mRNA has been
found in plants, Drosophila, and mammals. Requirements are perfect complementarity and
catalytically active Argonaute (Slicer).
Messenger RNA cleavage
Crystal structure of the Argonaute protein with siRNA (red) and mRNA (green) inserted by model building. RNA-binding PAZ domain, blue; nuclease PIWI domain, purple.
RNASILENCING
Tetrahymena
PlantFruit flyFission yeast
Transcriptional Gene Silencing (TGS)CONTROL OF THE CHROMATIN STATESmall noncoding RNAs silence expression of genes with homologous sequences by preventing transcriptionat the DNA. TGS inhibits gene transcription by forming heterochromatin or promoting methylation.
Fruit fly
Spreading A PRIMITIVE “IMMUNE SYSTEM”RNA silencing can spread from cell to cell in plants and confer a sort of plant-wide immunity to viruses. In the nematode C. elegans, silencing can be transmitted from generation to generation.
Plant Nematode
Plant
Plant Nematode Mouse Fruit fly
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Viral RNAViruses can produce RNAsto hijack the RNA silencing pathways in the infected cell, furthering the viruses’ survival.
Scientist-supplied RNAExogenous RNA can be taken up intocells where it interferes with geneexpression through the endogenoussilencing pathways. This technique hasproven to be a powerful method ofinhibiting and testing gene function inmany organisms.
MicroRNAs (miRNAs)After transcription by RNA poly-merase, the folded pri-miRNA isprocessed at the 5’ end by Drosha toproduce pre-miRNA. The pre-miRNA istranslocated into the cytoplasm byexportin-5, where Dicer will completeits maturation into miRNA.
RNA from repetitive DNAor aberrant RNA
In plants and nematodes, transposons and transgenes, which contain repetitive DNA, encode double-stranded RNA through bi-directional transcription or RdRp activity. Theresulting siRNAs are used in TGS to inhibit geneexpression from the original DNA. Centromericheterochromatin and the mating type locus in fission yeast are also silenced in this way.Aberrant RNA is similarly processed and eliminated.
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