Title: RNAi: Insight, Mechanisms and Potential
1RNAi Insight, Mechanisms and Potential
- Nobel Prize in Physiology or Medicine awarded to
- A. Fire and C. Mello
2What is RNA interference (RNAi)?
- The Process by which dsRNA silences gene
expression... - Degradation of mRNA or translation inhibition
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3What are sense and antisense RNA?
- Messenger RNA (mRNA) is single-stranded, called
"sense" because it results in a gene product
(protein).
5 C U U C A 3 mRNA3 G A A G U
5 Antisense RNA
4What are sense and antisense RNA?
- Antisense molecules interact with complementary
strands of nucleic acids, modifying expression of
genes.
5 C U U C A 3 mRNA3 G A A G U
5 Antisense RNA
5RNAi 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
6- 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
7RNAi like phenomena
- Plants
- Petunias
- Fungi
- Neurospora
- Animals
- Caenorhabditis elegans
8Alternate terms to RNAi
- PTGS (Posttranscriptional Gene Silencing)
- Cosuppression
- Quelling
- Virus-induced gene silencing
91990-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.
10Overexpression 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 wild-type
petunias, which led the authors to hypothesize
that the introduced transgene was cosuppressing
the endogenous CHS gene.
http//www.scq.ubc.ca/?p265
111992-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."
121995-The worm
- Guo and Kemphues studied par-1 gene during
embryogenesis - The worm, C. elegans
- has a fixed lineage hypodermis, intestine,
gonads - asymmetric divisions
131995- The worm
- Guo and Kemphues first studied Par-1 gene mutants
- Division Asymmetric?symmetric
- P-granule distribution
14Guo and Kemphues, 1995
15Both the antisense and sense strands effectively
silenced
wildtype
Par-1 RNAi
16- 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.
17Antisense 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 cant call it antisense when sense works
as well
Montgomery (2006) RNA interference unraveling a
mystery
18Craig 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.
19Andrew Fire
- In 1991, A. Fire successfully targeted genes by
antisense constructs from transgenes. - Sense constructs also exhibited silencing
activity.
20Fire and Mello
- Their paths crossed
- Mutual interest on a gene called pie-1.
- Discuss what the sense and antisense preparations
have in common
21Fire and Mello
- did simple math
- Sense (-sense) ? 0 (interference)
22Fire 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
23Fire 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
241998-Fire et al. and Mello, Nature
251998-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 post-transcriptional
silencing is in place.
26Unc-22 (Uncoordinated 22)
- Codes for a non essential myofilament
- It is present several thousand copies/cell
27- 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."
28Injection for RNAi
- 6-10 adult hermaphrodites were injected with
0.5x106-1x106 molecules into each gonadal arm.
29Unc-22 phenotype
- 4-6 hours after injection, eggs collected.
- Screened for phenotypic changes
- twiching
Exon Size RNA Phenotype
Exon 21-22 742 Sense Antisense Senseantisense Wildtype Wildtype Twicher (100)
Exon 27 1033 Sense Antisense Senseantisense Wildtype Wildtype Twicher (100)
30Mex-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
31Mex-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 mex-3B no mex-3 RNA is
detected.
32RNAi 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.
33Quantitative Assays
34Other possibilities
- Senseantisense 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.
35Conclusions
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36Conclusions
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37- 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
38Nobel Prize in Physiology or Medicine 2006
39The Laureates
40Essense of Nobel Prize
- Mellos words after he had heard the news I
seem too young, and isnt 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 Mulliss 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
41RNAi studies in PubMed
Number of Publications
Years before 2007
42Ways to induce silent phenotypes
- Timmons and Fire showed that feeding dsRNA works!
- Reversible and gene-specific effects
43Ways 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
44Mechanisms revealed
- 25bp species of dsRNA found in plants with
co-suppression 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
45RNAi two phases
- Initiation
- Generation of mature siRNA or miRNA
- Execution
- Silencing of target gene
- Degradation or inhibition of translation
46RNAi illustrated ?
47How does RNAi work?
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48siRNA 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
49RNA 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
50siRNA design
Mittal, 2004
51Custom-made siRNAs
52siRNA libraries
- Generation of a feeding clone
Tuschl, 2003
53siRNA libraries
- Result 16 757 bacterial strains
- 86.3 of predicted genes with RNAi phenotypes
assigned
54Assayed 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
55Endogenous 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
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56More miRNAs
- 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
Years before 2007
57Defense 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.
58Mammalian RNAi
McManus and Sharp, 2002
59Getting Around the Problem
- siRNA (21-22nt) mediate mammalian RNAi
- Introducing siRNA instead of dsRNA prevents
non-specific effects
60Some 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
61Drugging the worms
- AGING, CANCER, NEURODEGENERATION, NEUROBIOLOGY
62Genome-wide Screens
63Early Embryonic Phenotypes
- Sister chromatid separation
- Asymmetry of division
- Cytokinesis
- Pace of development
- Centrosome attachment
- Osmotic integrity
- Passage through meiosis
- Entry into interphase
- Cortical dynamics
- Nuclear appearance
64Genome-wide RNAi
- A total of 19,075 genes were targeted by dsRNAs.
- More than 40,000 time-lapse microscopy
recordings, scored and annotated.
65Genome-wide RNAi
- Only 11 genes showed detectable RNAi phenotype
- Between 600-800 genes are required for early
embryogenesis.
66Systems 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.
67The Future Integrative Biology
- Tis true, Theres magic in the web of it.
- William Shakespeare (1602-4)
68Networks 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.
69Systems Biology ApproachThree networks in one
70Does it make sense to combine?
71The embryogenesis network
72- 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.
73(No Transcript)
741995-Guo Kemphues
751995-Guo Kemphues
76A 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.
77- 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)
78Links to endogenous dsRNA
791999-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.
801999-Hamilton Baulcombe
811999-Hamilton Baulcombe concluded
82Discovery Project
83C. Elegans as a Disease Model
- AGING, NEURODEGENERATION, NEUROBIOLOGY
- Longevity model daf-2 mutants and RNAi showed
that reduced insuling signaling increases
resistance ox
84Discovery 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 3UTR Lee et al., 1993 - Required for negative regulation of lin-14
- lin-4 binds these sites
85More 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
86RNAi 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
87RNAi in other genes
- Unc-54 encodes a muscle myosin class II heavy
chain (MHC B) required for locomotion and
egg-laying
88Defense 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.
89Rethinking 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.