Transcript RNAi: Insight, Mechanisms and Potential

RNAi: Insight, Mechanisms
and Potential
Nobel Prize in Physiology or
Medicine awarded to
A. Fire and C. Mello
What is RNA interference (RNAi)?
“The Process by which dsRNA silences
gene expression...”
Degradation of mRNA or translation
inhibition
www.nobelprize.org
What are sense and antisense
RNA?
Messenger
RNA (mRNA)
is singlestranded,
called "sense"
because it
results in a
gene product
(protein).
5´
3´
C U U C A
G A A G U
3´
5´
mRNA
Antisense RNA
What are sense and antisense
RNA?
Antisense
5´
molecules
3´
interact with
complementary
strands of
nucleic acids,
modifying
expression of
genes.
C U U C A
G A A G U
3´
5´
mRNA
Antisense RNA
RNAi terms
dsRNA: double stranded RNA, longer than
30 nt
miRNA: microRNA, 21-25 nt.
– Encoded by endogenous genes
siRNA: small-interfering RNA, 21-25 nt.
– Mostly exogenous origin
Nature, it seems, is the popular game
for milliards, and milliards, and milliards
of particles playing their infinite game
of billiards and billiards and billiards
Piet Hein (1966) *
Piet Hein (December 16, 1905 - April 17, 1996), a Danish scientist, mathematician,
inventor, author, and poet; known for his short poems in the form of grook.
A grook ("gruk“, short for "GRin & sUK" ("laugh & sigh" in Danish) is a form of short
aphoristic poem.
*From ‘Critical Mass: How One Thing Leads To Another’ by Phillip Ball
RNAi like phenomena
Plants
– Petunias
Fungi
– Neurospora
Animals
– Caenorhabditis elegans
Alternate terms to RNAi
PTGS (Posttranscriptional Gene
Silencing)
Cosuppression
Quelling
Virus-induced gene silencing
1990-Petunias
Napoli et al. defined an RNAi-like
phenomenon and called it
“cosupression.”
chalcone synthase (CHS), a key enzyme
in flavonoid biosynthesis, the rate-limiting
enzyme in anthocyanin biosynthesis,
responsible for the purple coloration.
Overexpression of chalcone
synthase in petunias unexpectedly
resulted in white petunias
The levels of endogenous as
well as introduced CHS were
50-fold lower than in wildtype petunias, which led the
authors to hypothesize that
the introduced transgene
was “cosuppressing” the
endogenous CHS gene.
http://www.scq.ubc.ca/?p=265
1992-The mold
A rosette of the asci
Carlo Cogoni and Guiseppe Macino of the
Università di Roma La Sapienza in Italy
introduced a gene needed for carotenoid
synthesis in the mold Neurospora crassa:
– The introduced gene led to inactivation of the
mold's own gene in about 30% of the
transformed cells. They called this gene
inactivation "quelling."
1995-The worm
Guo and Kemphues studied par-1 gene
during embryogenesis
The worm, C. elegans
– has a fixed lineage: hypodermis, intestine,
gonads
– asymmetric divisions
1995- The worm
Guo and Kemphues
first studied Par-1
gene mutants
– Division:
Asymmetricsymmetric
– P-granule distribution
Guo and Kemphues, 1995
Both the antisense and sense
strands effectively silenced
wildtype
Par-1 RNAi
The reasonings about the wonderful and
intricate operations of Nature are so full of
uncertainty, that, as the Wise-man truly
observes, hardly do we guess aright at the
things that are upon earth, and with labour
do we find the things that are before us.
Stephen Hales (1727)
Stephen Hales (September 17, 1677 - January 4, 1761) was an English physiologist,
chemist and inventor; studied the role of air and water in the maintenance of both plant
and animal life.
‘Antisense’ Technology?
Sense RNA silences yet no hybridization of
sense RNA with sense mRNA is expected!
Intronic and promoter sequences do not silence.
ssDNA or dsDNA does not work!
Craig Mello at the Worm Meeting in Madison,
Wisconsin coined the term ‘RNAi’ and said that:
– “ We can’t call it ‘antisense’ when ‘sense’ works as
well”*
*Montgomery (2006) RNA interference: unraveling a mystery
Craig Mello
In 1996, C. Mello and his student S.
Driver also reported that sense RNAs
mimic antisense phenotype.
– Injection is made into a single site yet acts
more systemically.
Andrew Fire
In 1991, A. Fire successfully targeted
genes by antisense constructs from
transgenes.
Sense constructs also exhibited silencing
activity.
Fire and Mello
Their paths crossed:
– Mutual interest on a gene called pie-1.
– Discuss what the sense and antisense
preparations have in common
Fire and Mello
did simple math…
Sense + (-sense)  0 (interference)
Fire & Mello hypothesize on how
sense works?
ssRNA in vitro synthesized by bacteriophage
RNA pol might be contaminated by dsRNA
Bacteriophage RNA polymerase produce random
ectopic transcripts
Fire & Mello hypothesize on how
sense works?
DNA transgene arrays produce aberrant
RNA some with dsRNA character
certain repeated DNA sequences might be
expected to produce aberrant RNAs that would
then be capable of producing a silencing response
1998-Fire et al. and Mello, Nature
1998-Fire et al and Mello
Gel-purified ssRNA
Used purified ssRNA (antisense and
sense) separately and also together.
Tested ssRNA against different genes for
specificity
Tested whether a general posttranscriptional silencing is in place.
Unc-22 (Uncoordinated 22)
Codes for a non essential myofilament
It is present several thousand copies/cell
[If] There is some precision, there is some
science.
Herbert Spencer (1880)
Herbert Spencer (27 April 1820 – 8 December 1903) was an English philosopher and
prominent classic-liberal political theorist. Spencer analyzed human societies as
evolving systems, and coined the term "survival of the fittest."
Injection for RNAi
6-10 adult hermaphrodites were injected
with 0.5x106-1x106 molecules into each
gonadal arm.
Unc-22 phenotype
4-6 hours after injection, eggs
collected.
Screened for phenotypic
changes
– twiching
Exon
Size
RNA
Phenotype
Exon 21-22
742
Sense
Antisense
Sense+antisense
Wildtype
Wildtype
Twicher (100%)
Exon 27
1033
Sense
Antisense
Sense+antisense
Wildtype
Wildtype
Twicher (100%)
Mex-3
mex-3 encodes two RNA binding proteins;
in the early embryo, maternally provided
Mex-3 is required for specifying the
identities of the anterior AB blastomere and
its descendants, as well as for the identity
of the P3 blastomere and proper
segregation of the germline P granules
Mex-3 RNAi
b, Embryo from uninjected parent
(showing normal pattern of
endogenous mex-3 RNA20).
c, Embryo from a parent injected
with purified mex-3B antisense
RNA. Retain the mex-3 mRNA,
although levels may be somewhat
less than wild type.
d, Embryo from a parent injected
with dsRNA corresponding to mex3B; no mex-3 RNA is detected.
RNAi concentration and dose
response
3.6x106 molecules/gonad
– Sense phenocopied 1% of progeny
– Antisense phenocopied 11% of progeny
– dsRNA phenocopies 100% progeny and at
even 3x108 molecules/gonad.
Quantitative Assays
Other possibilities
Sense+antisense in low salt
Rapid sequential injection of sense &
antisense
– Both cause interference
– 1 hour apart injection of sense and antisense
leads to reduction in interference.
Conclusions
www.nobelprize.org
Conclusions
www.nobelprize.org
Nobody should be rich but those who
understand it
Wolfgang von Goethe
Johann Wolfgang Goethe (28 August 1749 – 22 March 1832), a German polymath:
a poet, novelist, dramatist, humanist, scientist, theorist, painter; the author of Faust
and Theory of Colours
Nobel Prize in Physiology or
Medicine 2006
Andrew Fire
Craig Mello
The Laureates
Essense of Nobel Prize
Mello’s words after he had heard the news: “I seem too
young, and isn’t the gap unusually short?”
Most Nobel prizes are given many years after the
relevant discovery. The Fire and Mello award, given just
eight years after publication of their paper, is reminiscent
of Kary Mullis’s 1993 chemistry Nobel. That prize was
awarded for his 1985 invention of the polymerase chain
reaction — a method of gene amplification that invaded
research labs just as fast and comprehensively as the
RNAi technique has.
Nature, News. Allison Abbott
Number of Publications
RNAi studies in PubMed
500
450
400
350
300
250
200
150
100
50
0
RNAi
siRNA
1
2
3
5
Years before 2007
10
Ways to induce silent phenotypes
Timmons and Fire showed that feeding dsRNA works!
Reversible and gene-specific effects…
Ways to induce silent phenotypes
Tabarra, Grishok, and Mello in 1998 demonstrated that
soaking in dsRNA also works!
Nomarski image showing embryos
produced by a wild-type mother
treated with pos-1 RNAi by
soaking. All except one embryo
(arrow) show the distinctive pos-1
embryonic arrest with no gut, no
body morphogenesis, and extra
hypodermal cells
pos-1 encodes a CCCH-type zinc-finger protein; maternally
provided POS-1 is essential for proper fate specification;
Mechanisms revealed
25bp species of dsRNA found in plants with cosuppression [Hamilton and Baulcombe, 1999]
Sequence similar to gene being suppressed
Drosophila: long dsRNA “triggers” processed
into 21-25bp fragments [Elbashir et al., 2001]
– Fragments = short interfering RNA (siRNA)
– siRNA necessary for degradation of target
RNAi: two phases
Initiation
– Generation of mature siRNA or miRNA
Execution
– Silencing of target gene
– Degradation or inhibition of translation
RNAi illustrated 
How does RNAi work?
www.nobelprize.org
siRNA biogenesis
Dicer (type III RNAse III) cleaves long dsRNA
into siRNA 21-25nt dsRNA from exogenous
sources
– Symmetric 2nt 3’ overhangs, 5’ phosphate groups
– Evidence for amplification in C. elegans and plants
RNA Induced Silencing Complex
(RISC)
RNAi effector complex
Preferentially incorporates one strand of
unwound RNA [Khvorova et al., 2003]
– Antisense
How does it know which is which?
– The strand with less 5’ stability usually incorporated
into RISC [Schwarz et al., 2003]
siRNA design
Mittal, 2004
Custom-made siRNAs
siRNA libraries
Generation of a feeding clone
Tuschl, 2003
siRNA libraries
Result: 16 757 bacterial strains
86.3% of predicted genes with RNAi
phenotypes assigned
Assayed Phenotypes: Examples
Emb – embryonic lethal
Ste – sterile
Gro – slow growth
Adl – adult lethal
Lvl – larval lethality
Lva – larval arrest
Bmd – body
morphological defects
Unc – uncoordinated
Clr – clear
Prz – paralyzed
Lon – long
Mlt – moulting defects
Egl – egg laying defects
Him – high incidence of
males
Endogenous RNAi-miRNA
We have hundreds of
different genes that
encode small RNA
(collectively, microRNA)
whose precursors can
form double-stranded
RNA. These can
activate the RNA
interference process and
thus switch off the
activity of various genes
with matching segments.
First miRNA is lin-4
www.nobelprize.org
No other miRNAs
found for 7 years!
– Second miRNA – let-7
[Reinhart et al., 2000]
– Non coding, 21nt RNA
– Regulates lin-14 in
same way as lin-4
Number of Publications
More miRNAs
70
60
50
40
miRNA
30
20
10
0
1
2
3
5
Years before 2007
10
Defense Against Viruses
www.nobelprize.org
Indeed, Baulcombe, Vance, and others have shown that, in the
continuing evolutionary war to survive and reproduce, plant viruses have
evolved genes that enable them to suppress silencing.
Mammalian RNAi
McManus and Sharp, 2002
Getting Around the Problem
siRNA (21-22nt) mediate mammalian
RNAi
– Introducing siRNA instead of dsRNA prevents
non-specific effects
Some applications of RNAi
Therapy
– Candidate genes, drug discovery, and therapy
Genome-wide RNAi screens
– Gene function
– Candidate genes and drug discovery
Systems biology
– Models of molecular machines
Drugging the worms
AGING, CANCER, NEURODEGENERATION,
NEUROBIOLOGY
Genome-wide Screens
Early Embryonic Phenotypes
Osmotic integrity
Passage through
meiosis
Entry into interphase
Cortical dynamics
Nuclear appearance
Sister chromatid
separation
Asymmetry of division
Cytokinesis
Pace of development
Centrosome attachment
Genome-wide RNAi
A total of 19,075 genes were targeted by
dsRNAs.
More than 40,000 time-lapse microscopy
recordings, scored and annotated.
Genome-wide RNAi
Only 11% genes showed detectable RNAi
phenotype
Between 600-800 genes are required for early
embryogenesis.
Systems Biology and RNAi
Cellular systems act as networks of interacting
components (genes, RNA, protein,
metabolites,…).
Genome-wide RNAi screens offers the potential
for revealing functions of each protein.
Combining RNAi screen data with other
highthroughput data (e.g., protein-protein
interaction, mRNA expression profiling) leads to
understanding of the organization of the cell
system.
The Future: Integrative Biology
‘Tis true, There’s magic in the web of it.
William Shakespeare (1602-4)
Networks of Early Embryogenesis
Protein-protein interaction dataset: binary physical
interactions between 3,848 C. elegans proteins
Transcriptome dataset: expression profiling similarity
above a given threshold among genes in the network
Phenotypic dataset: phenotypic similarity above
another threshold of 661 early embryogenesis genes.
RNA interference (RNAi) phenotypic signature consisting
of a vector describing specific cellular defects in early
embryogenesis.
Systems Biology Approach:
Three networks in one
Does it make sense to combine?
The embryogenesis network
The aesthetics of natural science and
mathematics is at one with the aesthetics
of music and paintings both inhere in the
discovery of a partially concealed pattern.
Herbert Simon (1996)
Herbert Alexander Simon (June 15, 1916 – February 9, 2001), an American political
scientist and polymath; one of founding fathers of Artificial Intelligence, information
processing, decision-making, and complex systems, the first to analyze the
architecture of complexity and to propose a preferential attachment mechanism to
explain power law distributions.
1995-Guo & Kemphues
1995-Guo & Kemphues
A book: Critical Mass by Phillip Ball
How one thing leads to other
He details the development
of key concepts in
contemporary physics, such
as self-organization, and
chaos; Next, he shows how
social scientists apply these
concepts to the study of
human organization.
the relationship between
global phenomena and local
actions.
The reader will appreciate the orderliness
of the lines… and he will see how this
orderliness points to the existence of a
fundamental governing principle.
George Kingley Ziph (1949)
Links to endogenous dsRNA
1999-Hamilton & Baulcombe
Hypothesis:
Antisense RNA
complementary to
the target mRNA
may be too short to
observe so is easy to
miss.
Tomato lines
transformed with
ACO cDNA under
the 35S promoter.
1999-Hamilton & Baulcombe
1999-Hamilton & Baulcombe
concluded:
Discovery Project
C. Elegans as a Disease Model
AGING, NEURODEGENERATION, NEUROBIOLOGY
– Longevity model: daf-2 mutants and RNAi showed that reduced insuling signaling
increases resistance ox
Discovery of miRNA
Discovery of the first miRNA, lin-4
– Non-coding, 22nt RNA
– Identified in screen for defects in timing of larval development
lin-4 mutation – ectopic larval stage 1-like cell divisions
at later stages
lin-14 mutations – reciprocal phenotype, same regulatory
pathway as lin-4
– lin-4 negatively regulates lin-14 translation
– lin-4 partially complementary to conserved sites in lin-14 3’UTR
[Lee et al., 1993]
– Required for negative regulation of lin-14
– lin-4 binds these sites
More miRNAs
miRNA is in other organisms?
– Let-7 Homologs were easily detected
[Pasquinelli et al., 2000]
Drosophila, sea urchins, mice, humans...
Indicates RNAi general conserved mechanism
RNAi in other genes
Hlh-1: myogenic regulatory factor (MRF)
subgroup of bHLH proteins; HLH-1 activity is
required during embryonic development for the
proper differentiation and function of body wall
muscle cells
RNAi in other genes
Unc-54: encodes a muscle myosin class II heavy chain
(MHC B) required for locomotion and egg-laying
Defense against transposons
RNAi may also help keep the transposable
elements that litter genomes from jumping
around and causing harmful mutations.
Plasterk's team and Mello, Fire, and their
colleagues found that mutations that
knocked out RNAi in C. elegans led to
abnormal transposon movements.
Rethinking of the antisense dogma
I have endeavored to show that it is the
peculiar function of physical science to
lead us to the confines of the
comprehensible, and to bid us behold it
and receive it in faith, till such time as the
mystery shall open.
James Clerk Maxwell (1856)
When 19th century Scottish physicist James Clerk Maxwell was faced with predicting the behavior
of gases made up of trillions of rapidly moving molecules, he borrowed from social scientists the
mathematical tools of statistics and successfully applied them to the problem.