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Title: RNAi: Insight, Mechanisms and Potential


1
RNAi Insight, Mechanisms and Potential
  • Nobel Prize in Physiology or Medicine awarded to
  • A. Fire and C. Mello

2
What is RNA interference (RNAi)?
  • The Process by which dsRNA silences gene
    expression...
  • Degradation of mRNA or translation inhibition

www.nobelprize.org
3
What 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
4
What 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
5
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

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
7
RNAi like phenomena
  • Plants
  • Petunias
  • Fungi
  • Neurospora
  • Animals
  • Caenorhabditis elegans

8
Alternate terms to RNAi
  • PTGS (Posttranscriptional Gene Silencing)
  • Cosuppression
  • Quelling
  • Virus-induced gene silencing

9
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.

10
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 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
11
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."

12
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

13
1995- The worm
  • Guo and Kemphues first studied Par-1 gene mutants
  • Division Asymmetric?symmetric
  • P-granule distribution

14
Guo and Kemphues, 1995
15
Both 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.
17
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 cant call it antisense when sense works
    as well

Montgomery (2006) RNA interference unraveling a
mystery
18
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.

19
Andrew Fire
  • In 1991, A. Fire successfully targeted genes by
    antisense constructs from transgenes.
  • Sense constructs also exhibited silencing
    activity.

20
Fire and Mello
  • Their paths crossed
  • Mutual interest on a gene called pie-1.
  • Discuss what the sense and antisense preparations
    have in common

21
Fire and Mello
  • did simple math
  • Sense (-sense) ? 0 (interference)

22
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

23
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

24
1998-Fire et al. and Mello, Nature
25
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 post-transcriptional
    silencing is in place.

26
Unc-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."
28
Injection for RNAi
  • 6-10 adult hermaphrodites were injected with
    0.5x106-1x106 molecules into each gonadal arm.

29
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 Senseantisense Wildtype Wildtype Twicher (100)
Exon 27 1033 Sense Antisense Senseantisense Wildtype Wildtype Twicher (100)
30
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

31
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 mex-3B no mex-3 RNA is
detected.
32
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.

33
Quantitative Assays
34
Other 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.

35
Conclusions
www.nobelprize.org
36
Conclusions
www.nobelprize.org
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
38
Nobel Prize in Physiology or Medicine 2006
  • Andrew Fire
  • Craig Mello

39
The Laureates
40
Essense 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

41
RNAi studies in PubMed
Number of Publications
Years before 2007
42
Ways to induce silent phenotypes
  • Timmons and Fire showed that feeding dsRNA works!
  • Reversible and gene-specific effects

43
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
44
Mechanisms 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

45
RNAi two phases
  • Initiation
  • Generation of mature siRNA or miRNA
  • Execution
  • Silencing of target gene
  • Degradation or inhibition of translation

46
RNAi illustrated ?
47
How does RNAi work?
www.nobelprize.org
48
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

49
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

50
siRNA design
Mittal, 2004
51
Custom-made siRNAs
52
siRNA libraries
  • Generation of a feeding clone

Tuschl, 2003
53
siRNA libraries
  • Result 16 757 bacterial strains
  • 86.3 of predicted genes with RNAi phenotypes
    assigned

54
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

55
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
56
More 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
57
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.

58
Mammalian RNAi
McManus and Sharp, 2002
59
Getting Around the Problem
  • siRNA (21-22nt) mediate mammalian RNAi
  • Introducing siRNA instead of dsRNA prevents
    non-specific effects

60
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

61
Drugging the worms
  • AGING, CANCER, NEURODEGENERATION, NEUROBIOLOGY

62
Genome-wide Screens
63
Early 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

64
Genome-wide RNAi
  • A total of 19,075 genes were targeted by dsRNAs.
  • More than 40,000 time-lapse microscopy
    recordings, scored and annotated.

65
Genome-wide RNAi
  • Only 11 genes showed detectable RNAi phenotype
  • Between 600-800 genes are required for early
    embryogenesis.

66
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.

67
The Future Integrative Biology
  • Tis true, Theres magic in the web of it.
  • William Shakespeare (1602-4)

68
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.

69
Systems Biology ApproachThree networks in one
70
Does it make sense to combine?
71
The 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)
74
1995-Guo Kemphues
75
1995-Guo Kemphues
76
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.

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)

78
Links to endogenous dsRNA
79
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.

80
1999-Hamilton Baulcombe
81
1999-Hamilton Baulcombe concluded
82
Discovery Project
83
C. Elegans as a Disease Model
  • AGING, NEURODEGENERATION, NEUROBIOLOGY
  • Longevity model daf-2 mutants and RNAi showed
    that reduced insuling signaling increases
    resistance ox

84
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 3UTR Lee et al., 1993
  • Required for negative regulation of lin-14
  • lin-4 binds these sites

85
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

86
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

87
RNAi in other genes
  • Unc-54 encodes a muscle myosin class II heavy
    chain (MHC B) required for locomotion and
    egg-laying

88
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.

89
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.
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