RNAi: Insight, Mechanisms and Potential

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RNAi Insight, Mechanisms and Potential

• Nobel Prize in Physiology or Medicine awarded to
• A. Fire and C. Mello

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What is RNA interference (RNAi)?

• The Process by which dsRNA silences gene
  expression...
• Degradation of mRNA or translation inhibition

www.nobelprize.org
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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
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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
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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

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• 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
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RNAi like phenomena

• Plants
• Petunias
• Fungi
• Neurospora
• Animals
• Caenorhabditis elegans

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Alternate terms to RNAi

• PTGS (Posttranscriptional Gene Silencing)
• Cosuppression
• Quelling
• Virus-induced gene silencing

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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.

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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
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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."

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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

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1995- The worm

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

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Guo and Kemphues, 1995
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Both the antisense and sense strands effectively
silenced
wildtype
Par-1 RNAi
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• 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.
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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
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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.

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Andrew Fire

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

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Fire and Mello

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

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Fire and Mello

• did simple math
• Sense (-sense) ? 0 (interference)

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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

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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

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1998-Fire et al. and Mello, Nature
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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.

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Unc-22 (Uncoordinated 22)

• Codes for a non essential myofilament
• It is present several thousand copies/cell

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• 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."
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Injection for RNAi

• 6-10 adult hermaphrodites were injected with
  0.5x106-1x106 molecules into each gonadal arm.

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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)
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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

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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.
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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.

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Quantitative Assays
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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.

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Conclusions
www.nobelprize.org
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Conclusions
www.nobelprize.org
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• 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
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Nobel Prize in Physiology or Medicine 2006

• Andrew Fire
• Craig Mello

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The Laureates
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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

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RNAi studies in PubMed
Number of Publications
Years before 2007
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Ways to induce silent phenotypes

• Timmons and Fire showed that feeding dsRNA works!
• Reversible and gene-specific effects

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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
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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

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RNAi two phases

• Initiation
• Generation of mature siRNA or miRNA
• Execution
• Silencing of target gene
• Degradation or inhibition of translation

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RNAi illustrated ?
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How does RNAi work?
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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

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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

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siRNA design
Mittal, 2004
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Custom-made siRNAs
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siRNA libraries

• Generation of a feeding clone

Tuschl, 2003
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siRNA libraries

• Result 16 757 bacterial strains
• 86.3 of predicted genes with RNAi phenotypes
  assigned

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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

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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

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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
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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.

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Mammalian RNAi
McManus and Sharp, 2002
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Getting Around the Problem

• siRNA (21-22nt) mediate mammalian RNAi
• Introducing siRNA instead of dsRNA prevents
  non-specific effects

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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

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Drugging the worms

• AGING, CANCER, NEURODEGENERATION, NEUROBIOLOGY

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Genome-wide Screens
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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

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Genome-wide RNAi

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

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Genome-wide RNAi

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

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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.

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The Future Integrative Biology

• Tis true, Theres magic in the web of it.
• William Shakespeare (1602-4)

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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.

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Systems Biology ApproachThree networks in one
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Does it make sense to combine?
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The embryogenesis network
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• 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.
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(No Transcript)
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1995-Guo Kemphues
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1995-Guo Kemphues
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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.

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• 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)

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Links to endogenous dsRNA
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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.

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1999-Hamilton Baulcombe
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1999-Hamilton Baulcombe concluded
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Discovery Project
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C. Elegans as a Disease Model

• AGING, NEURODEGENERATION, NEUROBIOLOGY
• Longevity model daf-2 mutants and RNAi showed
  that reduced insuling signaling increases
  resistance ox

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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

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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

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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

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RNAi in other genes

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

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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.

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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.