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Regulation of gene expression by
small RNAs
; Discovery of small RNAs and their biogenesis and functions
Yoontae Lee
POSTECH
Central Dogma of Molecular Biology
By Francis Crick in1958
Expanded Central Dogma
Howard Temin and
David Baltimore
in 1970
How
muchdo
dowe
we know
know about
ourour
genome?
How
much
about
genome?
Genome
Known function (known genes) 3%
• 3 billion bp
• ~30,000 genes
Unknown function
97%
Junk ?
Supporting structure of the
genome?
Transcriptome
2%
Coding RNAs (exon)
Non-coding RNAs
(including introns)
98%
(Mattick, 2001)
Non-coding RNA studies are important to better understand our genome!!!
How much
do we know
about
our genome?
Non-coding
RNA
(ncRNA)
1. Transfer RNA (tRNA)
2. Ribosomal RNA (rRNA)
3. Small nuclear RNA (snRNA)
4. Small nucleolar RNA (snoRNA)
5. Long ncRNA (ex. Xist, Tsix, lincRNA…)
6. Small ncRNA (ex. siRNA, microRNA, piRNA…)
……..
Historical view of Small RNA discovery
Discovery of PTGS
• First discovered in plants (1990)
• Introduction of a transgene homologous
to an endogenous gene results in both
genes being suppressed!
• Also called Co-suppression
Chalcon synthase
Richard
Jorgensen
Richard
Jorgensen
Co-suppression
Quelling in Neurospora crassa
• In 1992, Romano and Macino discovered this phenomenon.
• albino-1 (al-1) gene, needed for the biosynthesis of the caroten
oids, which confer to N. crassa its typical orange pigmentation.
• Introducing extra al-1 copies within the N. crassa genome resul
ts in about 30% of colonies displaying a white phenotype, which
is identical to the one of al-1 mutants.
• This phenomenon was termed quelling.
Historical view of Small RNA discovery
RNA interference (RNAi)
Small RNA mediated target RNA cleavage,
post-transcriptional gene regulatory mechanism.
Antisense Technology
• Antisense technology has been used for ~20 years
• Introduction of an antisense gene (or antisense RNA)
into cells or organisms to block translation of the
sense mRNA.
• Alternative to gene knock-outs, which are very
difficult to do in higher plants and animals.
• The “antisense effect” was probably due to RNAi
rather than inhibiting translation.
Dr. Fire’s idea!!
In vitro transcription reaction
could produce unexpected
RNA transcripts as well!!
Gel purified sense or
antisense RNA
transcripts failed to
suppress the gene
expression.
Discovery of RNA interference (RNAi)
(Nature 1998)
Andrew Fire
unc-22
unc-22
Craig Mello
unc-22
Novel Prize in 2006
The phenotype found in unc-22 KO worms
Discovery of small RNAs generated from transgenes
Tomato lines transformed with a tomato 1aminocyclopropane-1-carboxylate oxidase (ACO)
cDNA sequence that places downstream of the
cauliflower mosaic virus 35S promoter.
T5.2 and T5.3 lines show PTGS of the ACO transgene
and express small RNAs.
Production of 21nt small RNAs from long dsRNA substrates
Incubation of radiolabelled long dsRNA in
Drosophila embryo lysate.
Small interfering RNA (siRNA) mediates RNAi.
Thomas Tuschl
RNase III enzyme Dicer generates siRNAs from long dsRNA
2001 Nature
Long dsRNA
~21nt siRNA
Gregory Hannon
How RNAi works
~20nt
PTGS in Plants and Neurospora
Overexpression of transgene
RNA dependent RNA polymerase
(Baulcombe 2004 Nature)
Knocking-down of a specific gene expression in C. elegans
using RNAi
Knocking-down of a specific gene expression in Drosophila
using RNAi
~500 bp
(Springer Japan KK. 2008)
Knocking-down of a specific gene expression in plants using
RNAi
Cauliflower
Mosaic Virus
(CaMV) 35S
promoter
MMG 445 Basic Biotech. 2009
Historical view of Small RNA discovery
Long dsRNA causes interferon response in mammalian cells.
Introduction of long
dsRNA into mammalian
cells induces interferon
response.
Long dsRNA can not be
used for knock-down
of specific gene
expression in
mammalian cells.
Honda and Taniguchi Nature Reviews Immunology 6, 644–658 (September 2006) | doi:10.1038/nri1900
2001 Nature
21nt siRNA
Thomas Tuschl
How does Dicer recognize, measure and cleave long dsRNA?
• PAZ domain recognizes 2nt
overhang at the 3’end of dsRNA.
• RNaseIII domains cleave
dsRNA at the 21st nt from the
3’end.
Giardia Dicer
Requirement of 2nt 3’ overhang for efficient dicer processing
61mer duplex
In vitro dsRNA processing assay
using recombinant dicer protein
(RNA 2006)
Nature 2011
siRNA pathway
induced
Argonaute (Ago) is a
core protein of the RISC.
Ago proteins in various organisms
In human and
mouse, there are
4 Ago proteins
(Ago1~4).
Structure of Ago protein
siRNA
Target
mRNA
Ago2 has a “slicer” activity.
P32
Luc target transcript
siRNA against Luc mRNA
5’
3’
Ago
FLAG-HA
Cleaved Luc transcript
(Gunter et al. 2004 Mol Cell)
siRNA guided cleavage of target mRNAs by RISC
Target RNAs
siRNA
11th
•Target RNA cleavage occurs
at the phosphodiester bond
between the bases
complementary to the 10th
and 11th bases of the siRNA.
10th
Martinez J , Tuschl T Genes Dev. 2004;18:975-980
Asymmetry in the Assembly of the RNAi Enzyme Complex
The relative stability of the base pairs
at the 5’ ends of the two siRNA strands
determine the degree to which strand
participates in the RNAi pathway.
(Dianne et al. 2003 Cell)
Which strand will be selected?
What is a microRNA (miRNA) ?
• A miRNA is defined as a single-stranded RNA of ~22 nt, which is
generated by the RNase III-type enzyme from an endogenous
transcript that contains a local hairpin structure.
Typical secondary structure of animal
miRNA precursors
MicroRNA biogenesis and action modes
•Pre-miRNA is processed into
miRNA duplex by the cytoplasmic
RNase III Dicer;
•A guide strand is selected from
miRNA duplex and loaded onto
argonaute protein to form
miRISC;
•miRISC binds to target mRNAs
to cause translational repression
or mRNA cleavage.
miRISC
Biogenesis and action mechanism
microRNA (miRNA)
Small interfering RNA (siRNA)
miRNA vs siRNA
• miRNAs differ from small interfering RNAs (siRNAs) in the origin.
miRNAs originate from hairpin-shaped precursors while siRNAs are
produced from long dsRNAs.
• Both miRNAs and siRNAs induce translational repression/mRNA
degradation and mRNA cleavage.
• Similar set of protein factors are involved in miRNA pathway and
siRNA pathway. In humans, the miRISC is indistinguishable from
the siRISC.
• In flies and plants, different homologues are often assigned to take
on different roles. For instance, fly AGO1 functions in the miRNA
pathway while fly AGO2 is critical in siRNA pathway.
Small RNA pathways in Drosophila
Two pathways exist
separately in flies.
miRNA mediated translational repression
By interfering with eIF4F-cap
recognition and 40S small
ribosomal subunit recruitment or
by antagonizing 60S subunit
joining and preventing 80S
ribosomal complex formation or
possibly by interfering with the
closed loop formation of mRNAs
mediated by the eIF4G-PABP
(because GW182 can bind to
PABP).
By inhibiting ribosome elongation,
inducing ribosome drop-off, or
facilitating proteolysis of nascent
polypeptides.
There is no mechanistic insight to
any of these proposed
“postinitiation block” models.
miRNA mediated mRNA decay
Poly A specific ribonuclease (PARN)
Poly A nuclease (PAN)
(3’-5’ exonuclease)
Xrn1
(5’-3’ exonuclease)
Following deadenylation, the 5’ cap is removed by the DCP1/DCP2 decaping enzymes.
Recognition of target mRNAs by miRNAs
•Positions 2-7 or 2-8 of miRNAs
form the critical “seed” for target
recognition;
• In case of the Drosophila miR-9
family, their sequences are identical
through position 8, but strongly
diverge beginning with position 9.
The members of this family are
inferred to have at least some
common targets because of their
shared seed, although their target
properties are likely somewhat
distinct because of their divergent 3'
ends.
7
2
mRNA
microRNA
(Reinhart et al., 2000, Nature)
Three members of the Drosophila miR-9 family
miRNA target prediction
Criteria to be considered for miRNA target prediction
1. The sequences in the 3’UTR of mRNAs that are perfectly matched with
the seed sequences of a particular miRNA.
2. Evolutionary conservation of the miRNA target sites.
3. Secondary RNA structures surrounding the miRNA target sites, which
may affect accessibility of miRISC to the target mRNA.
miRNA target prediction databases
1.
Targetscan : http://www.targetscan.org/
2.
PicTar : http://pictar.mdc-berlin.de/
3.
miRanda : http://www.microrna.org/microrna/home.do
4.
microCosm : http://www.ebi.ac.uk/enrightsrv/microcosm/htdocs/targets/v5/#
How many miRNA genes are there in each organism?
You can find it through miRBase website (http://www.mirbase.org/)
The number of miRNA genes in each
organism (as of 07/07/14)
Homo sapiens : 2588
Mus musculus :1915
Drosophila melanogaster : 466
Caenorhabditis elegans : 434
Arabidopsis thaliana :427
Expression of most genes could be regulated by miRNAs!!
Discovery of MicroRNA
1. Discovery of small temporal RNAs (stRNAs) – lin-4 and let-7
Victor Ambros
Gary Ruvkun
1993: The C. elegans heterochronic gene lin-4 encodes small RNAs with
antisense complementarity to lin-14 (Cell, V. Ambros lab., G. Ruvkun lab.).
lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
2000: The 21-nucleotide let-7 RNA regulates developmental timing in C.
elegans. (Nature, G. Ruvkun lab.).
The function of the lin-4 in the C. elegans seam-cell lineage
Lin-4 is induced at the transition
between the L1 and L2 larval stages
and this correlates with a decrease
in lin-14 protein levels.
In lin-4 mutants, lin-14 levels are not
downregulated at this stage.
Lin-4 mutant worms display
reiteration of L1 stage throughout
development.
(1993 Cell)
Discovery of MicroRNA
2. Classification of microRNAs (miRNAs)
Victor Ambros
Thomas Tuschl
David Bartel
2001: An abundant class of tiny RNAs, termed microRNAs, was identified
in C. elegans, Drosophila, and humans. (Science, V. Ambros lab., T. Tuschl
lab., D.P. Bartel lab.)
Cytoplasmic RNase III Dicer processes pre-miRNAs into mature
miRNAs.
(G&D 2001)
Ronald Plasterk
(Cell 2001)
Craig Mello
(Science 2001)
Philip Zamore
Genomic location and gene structure
• Some miRNA loci are found in non-protein coding transcription
units whereas some are located in protein coding genes.
• About half of the human miRNA loci are found in introns.
How are microRNAs generated?
Genomic organization of microRNA genes
(Lau et al., 2001, Science)
• Some miRNA genes are present in close conjunction.
• The expression profiles of the clustered miRNAs are similar.
Two possible modes of gene expression of
clustered microRNAs
Monocistronic
transcriptional unit ?
Polycistronic
transcriptional unit ?
Gene
Nascent
transcript
Mature
miRNA
Single step
processing ?
Multiple step
processing ?
Do long transcripts containing miRNAs exist
in cells?
Primary transcript of microRNAs
RT-PCR
from HeLa total RNA
There exist long transcripts
containing pre-miRNAs.
Stepwise processing from the long transcript ?
In vitro processing assay
293T cells
In vitro transcription
T7
NTP mix
32
P-UTP
T7 polymerase
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*
Uniformly labeled
miRNA precursor
Cell lysate
*
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*
**
*
*
*
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o
37 C, 90 min
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Denaturing polyacrylamide gel
Stepwise processing of microRNAs
(Pre-miR-30a)
(mature miR-30a)
The pri-miRNAs are processed
in a stepwise manner in vitro.
Compartmentalization of miRNA processing
Subcellular fractionation followed by
in vitro processing
• The first step occurs
mainly in the nucleus.
• The second step is
confined in the cytoplasm.
Model for
microRNA biogenesis
(Lee et al., EMBO, 2002)
• Maturation of miRNA occurs
through at least two steps.
• The two steps are
compartmentalized into the
nucleus and the cytoplasm.
• Pre-miRNAs may serve as
the substrate for nuclear
export.
• Regulation of miRNA
biogenesis may occur at
multiple levels.
?
• Which RNA
polymerase
transcribes miRNA
genes?
• What is the
enzyme for primiRNA processing
in the nucleus?
?
RNA polymerase II
transcribes miRNA genes.
RNA pol II
(Lee et al., EMBO, 2004)
?
• Pri-miRNAs have a cap
structure at 5’ end and polyA
tail at 3’ end.
• Treatment with a-amanitin
suppresses transcription of
miRNA genes.
•RNA pol II is physically
associated with miRNA gene
promoter.
RNA pol II
?
What is the nuclear
enzyme responsible
for pri-miRNA
processing?
RNase III type enzymes
• Require dsRNA structure;
• Generate 2-nt 3’ overhangs;
• Require divalent cations (Mg2+) for catalysis.
Domain organization
Dicer
L44
Drosha
Helicase
PAZ
RIII-a
RIII-b dsRBD
Subcellular localization
Cytoplasm
Mitochondria
RIII
RIII-a RIII-b dsRBD
Nucleus
In vitro processing of pri-miRNAs by Drosha
Expression of Drosha-FLAG in
293T cells
Immunopurification using antiFLAG antibody
In vitro processing
Drosha initiates primary
miRNA processing.
RNA?pol II
(Lee et al., Nature, 2003)
Drosha
• Stepwise processing is
mediated by two RNase III
proteins:
Drosha and Dicer.
RNA pol II
Drosha requires
cofactors for primiRNA processing?
Drosha
Gel exclusion chromatography of Drosha
Preperation of nuclear extracts
from 293T cells
Fractionation of nuclear proteins
using gel exclusion chromatography
In vitro pri-miRNA processing assay
& Western blot analysis
Human Drosha is part of ~650-kDa complex.
Partnership between RNase III and dsRBD protein
In Humans,
Dicer – TRBP
dsRBD
dsRBD
dsRBD
Dicer – PACT
dsRBD
dsRBD
dsRBD
dsRBD
dsRBD
miRNA accumulation,
RNAi process
In Drosophila,
Dicer-2 – R2D2
Dicer-1 – Loquacious/R3D1
dsRBD
Strand selection, RNAi
process
dsRBD
dsRBD
Pre-miRNA processing
In C. elegans,
Dicer – RDE4
dsRBD
dsRBD
siRNA accumulation,
RNAi process
In Plants,
DCL-1 – HYL1
dsRBD
dsRBD
miRNA accumulation
Identification of Drosha binding proteins
Transfection with Drosha-FLAG
expression plasmids to 293T cells
Immunoprecipitation of Drosha-FLAG using
anti-FLAG antibody conjugated beads
7.5% SDS-PAGE & MALDI-TOF
DGCR8
(DiGeorge syndrome critical region gene 8)
WW
dsRBD dsRBD
773 amino acids
Reconstitution of pri-miRNA processing
Immunoprecipitation of Drosha-FLAG
Detachment of Drosha interacting
proteins from immunoprecipitates
of Drosha-FLAG by harsh washing
with 2.5M NaCl buffer
Reconstitution of pri-miRNA
processing activity by adding GSTDGCR8 to immunoprecipitates of
Drosha-FLAG
DGCR8 is required for pri-miRNA processing.
Pri-miRNA processing
by Drosha-DGCR8
complex
RNA?pol II
(Han and Lee et al., Genes Dev., 2004)
DroshaDGCR8
• ~650-kDa protein complex
containing Drosha and
DGCR8 executes pri-miRNA
processing.
• Drosha and DGCR8 are
necessary and sufficient for
pri-miRNA processing.
Molecular basis for the
recognition of primary
miRNAs by the DroshaDGCR8 complex
(Han, Lee, and Yeom et al., Cell, 2006)
• DGCR8 anchors at ssRNAdsRNA junction of pri-miRNAs.
• Drosha cleaves pri-miRNAs at
11bp away from SD junction.
Nuclear export by exportin 5 (Exp5)
• Pre-miRNA is recognized and exported to the cytoplasm by
exportin 5 (Exp5).
• Exp5 belongs to the Ran-dependent nuclear transport receptor
family. Exp5 forms an export complex together with pre-miRNA
and a small GTPase, Ran.
Exportin 5 recognition motif
• Exp5 recognizes the ‘minihelix motif’ which comprises of a
stem of ~14 nt and a short 3’ overhang.
(Gwizdek et al., 2001)
Minihelix motif =
~14-nt stem + short 3’ overhang
• Pre-miRNAs are the major natural cargos for Exp5.
(Lund et al., 2003;
Lee et al., 2003)
• Artificial small hairpin RNAs (shRNAs) can be transported by Exp5.
(Yi et al., 2003;
Brummelkamp et al., 2002)
microRNA maturation
in animals
miRNA gene
Transcription
RNA polymerase II
V. Narry Kim
pri-miRNA
Cropping
Strand selection
& RISC
assembly
Drosha-DGCR8
(Microporcessor)
Export
pre-miRNA
Exportin 5
Nucleus
Cytoplasm
Dicing
Dicer
Ago2
TRBP
Mature
miRNA
in miRISC
Mirtron pathway
Alternative microRNA
biogenesis pathway that
bypasses nuclear Drosha
processing step.
After splicing, some introns
can form pre-miRNA-like
structure called mirtron.
Spliceosome
DroshaDGCR8
Spliceosome
A dicer-independent miRNA biogenesis
pathway that requires Ago catalysis
miR-451 : 5’ AAACCGUUACCAUUACUGAGUU 3’
Pre-miR-451 bypasses Dicer processing
but instead requires Ago2 catalysis for
its maturation.
miRNA biogenesis in plants
miRNA gene
Transcription
RNA polymerase II
pri-miRNA
Processing
Dicer-like 1 (DCL1)
HYL1
Export
Hasty
(HST)
Nucleus
Cytoplasm
Strand selection
& RISC assembly
AGO1
Mature
miRNA
in miRISC
Generation of
shRNAs
miRNA shRNA cassette shRNA cassette
(pol II)
(pol III)
gene
• Knowledge of miRNA
biogenesis is important not
only to understand the
biology of miRNAs but also
to improve RNAi technology.
• miRNA biogenesis factors
are required for production
of siRNAs from small
hairpin RNAs (shRNAs).
Mature miRNA
(miRISC)
siRNA
(siRISC)
Pol II driven shRNA expression vector
Thermo scientific open biosystem