Title: how this does it works in mature RISC
1how this does it works in mature RISC
The structural basis for binding of RNA to the
argonaute protein has been examined by X-ray
crystallography of the binding domain of an
RNA-bound argonaute protein. The phosphorylated
5' end of the RNA strand enters a conserved basic
surface pocket and makes contacts through a
divalent cation such as magnesium and by aromatic
stacking between the 5' nucleotide in the siRNA
and a conserved tyrosine residue. This site is
thought to form a nucleation site for the binding
of the siRNA to its mRNA target.2
2 Piante metazoi
- miRNA 3 end CH3 OH
- Localization of target sites in the coding
sequence in the 3UTR - Number of target 1 multipli
- Mechanism degradation translational repression
3Regulation of gene expression by small (21-nt)
RNAs
RNA interference (RNAi) pathway MicroRNA
pathway
Antisense genes Repetitive elements
MicroRNA precursors encoded in the genome
Transfection
Viral infection
dsRNA
DICER
DICER
siRNAs (20-bp)
RITS
RISC
RISC-like miRNP
mRNA
AAAA
3-UTR
mRNA degradation
Chromatin silencing
Translational repression
4MicroRNAs 21-nt regulators of gene expression
Drosha complex
RISC-like miRNPcomplex
Dicer
miRNA
(nucleus)
Ago
Pri-miRNA
Pre-miRNA
Ago
AAAAAAAAA
3-UTR
- Conserved from plants to humans
- 800 miRNAs may operate in primates
- Tissue- and development-specific expression
- 30 of all human genes are predicted to be
miRNA targets - Function as miRNPs, with Argonautes (Ago), FMRP,
Gemins
5Family of Argonaute proteins
Piwi
PAZ
900aa
Hiwi/Piwi
Ago proteins in different organisms
Mammals 4 Drosophila 5 C. elegans 27
Ago
Carmell Hannon, 2002
Ago is the slicer and releases the siRNA from the
dsRNA by its slicer activity
6miRNAs and their mRNA targets emerging patterns
Bartel et al., 2004
- Bind to mRNA 3-UTRs targets spread over
kilobases of 3-UTR - One miRNA can control activity of many different
mRNAs - A single mRNA can be controlled by more than one
miRNA
- Major advantages of miRNA regulation?
- Networking and fine-tuning of gene expression
- Rapid (and possibly reversible) repression
7- miRNAs major challenges
- Identification of mRNA targets
- Mechanism of inhibition of protein accumulation
From W. Filipowicz
8 How do miRNAs inhibit productive translation?
miRNP
- Observations
- Proteins do not accumulate (different metazoa)
- mRNA levels largely unaffected (C. elegans,
Drosophila, mammals) but - some recent papers (Lim et al., 2005
Pasquinelli et al., 2005) report - considerable mRNA degradation
- mRNA associated with polysomes (C. elegans
Olson Ambros, 99)
- Questions
- Is translation per se inhibited? Or proteins are
made but undergo - proteolysis?
- If translation is targeted, what is the
mechanism? - Contribution of mRNA degradation
- Do all miRNAs function in the same way?
From W. Filipowicz
9 mRNA reporters to study miRNA-mediated repression
- Reporters responding to the endogenous let-7
miRNP
From W. Filipowicz
10Tethering of human Argonautes to the reporter
mRNA mimics the effect of miRNAs
HA- hAgo2
N
HA- hAgo2
N
HA- LacZ
AAAAA
Rr-luc 5BoxB
BoxB hairpins
Reporter activity
Western (?-HA Ab)
From W. Filipowicz
11- Summary of tethering experiments
- All tethered hAgo proteins (hAgo2, 3 4)
inhibit - protein accumulation
- Inhibition occurs by miRNA-like mechanism
(several BoxB - required, no dependence on position in
3UTR, etc)
- Main function of miRNAs is to guide repressive
proteins - to the mRNA
- Integrity of hAgo2 is essential Ago as a
repressor?
AUG
From W. Filipowicz
12Inhibition of productive translation by
endogenous let-7 miRNP in HeLa cells
13Translational initiation as a target of repression
AAAAAA
14The miRNA-mediated repression targets
translational initiation
Polysomal distribution of RL mRNAs
Control is loaded onto polysomes
3xbuldge remains in RNPs
From W. Filipowicz
15RNA transfections to study repression by miRNAs
T7
- Questions
- Is capping essential?
- IRES versus cap
- Is polyA essential?
- Is nuclear history important?
Renilla
AAAAA
1X let7 perfect
AAAAA
Renilla
3X let7 bulged
16Transfected mRNAs are repressed by endogenous
let-7 RNA
Renilla reporter
Firefly reporter
RL
PolyA
FL
PolyA
PolyA-
PolyA-
RL (arbitrary units)
FL activity (arbitrary units)
Con
Perf
Con
Perf
3xBulge
3xBulge
1xBulge
From W. Filipowicz
17Cap-independent initiation is not inhibited by
miRNAs
- Identical results with Hepatitis C Virus (HCV)
IRES - When both IRES and capped mRNAs are transfected
together, - only capped mRNA is repressed
From W. Filipowicz
18Tethering of eIF-4E or eIF-4G does not respond to
miRNA repression
Yes
No
2 -BoxB
FL
RL
Let-7
95nt
75nt
LacZ
eIF4-E
eIF4-G
N-HA
N-HA
N-HA
FL cistron RL cistron
From W. Filipowicz
19In vitro translational repression by miRNAs -
let-7 miRNA inhibits cap-dependent, but not
cap-independent, translation in an ascites
extract. (A) Schematic structure of mRNAs
containing the EMCV IRES. (B) The EMCV IRES
confers resistance to repression of RL-6xB
translation. EMCV IRESs containing mRNAs were
translated for 1 hour in the presence or absence
of antilet-7 2'-O-Me oligonucleotides. (C)
ApppG-capped RL-6xB mRNA is refractory to
let-7mediated inhibition. (D) Effect of eIF4F
addition on miRNA-mediated repression. Extracts
were supplemented with the indicated amounts of
purified eIF4F, and Rluc activity was assayed
after a 1-hour translation reaction. Data are
depicted as a percentage of translation relative
to RL in the presence or absence of antilet-7
2'-O-Me oligonucleotide.
20Ago proteins localize to P-bodies in stable cell
lines and associate with P-body components
Control HeLa
NHA-Ago2
NHA-Ago3
HA-Ago2
A
B
C
D
HA (Ago2)
HA (Ago2)
HA (Ago2)
HA (Ago3)
Endog. Ago
a-Ago
a-HA
Lsm1
Xrn1
Xrn1
Dcp1a
E
Merge
Merge
Merge
Merge
F
HA-Ago2
NHA-Ago2
NHA-Ago3
Liu et al., 2005 Sen Blau, 2005