RNA interference: Little RNAs, Big Impact

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The Silent Revolution RNAi Biology, Reserch and
Therapeutics

• RNA interference Little RNAs, Big Impact
• Lecture 5.3
• Stephanie Minnema
• University of Calgary
• http//2d.bjmu.edu.cn

Juan Carlos Gallego-Gómez. PECET Sede de
Investigación Universitaria SIU-UdeA. Medellín
, June 21th /2007
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What Well Cover

• What is RNAi/ useful terms
• Brief history of RNAi
• Biogenesis and mechanisms of action
• Applying RNAi to model systems
• Endogenous RNAi miRNA in the genome
• New frontiers for RNA

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What is RNA Interference (RNAi)

• The Process by which dsRNA silences gene
  expression... Mittal, 2004
• Generally Post transcriptional level (PTGS)
• Degradation or translation inhibition
• Blazing hot topic...
• 2425 published articles this year!
• A field with many unknowns

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Handy RNAi Terms

• dsRNA double stranded RNA, longer than 30 nt
• miRNA microRNA, 19-25 nt.
• Encoded by endogenous genes.
• Hairpin precursors
• Recognize multiple targets.
• siRNA short-interfering RNA, 21-22 nt.
• Mostly exogenous origin.
• dsRNA precursors
• May be target specific

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An Arbitrary Distinction?

• miRNA vs. siRNA?
• Discovered in different ways
• Similar biogenesis
• Share common pathway components
• Common pathway outcomes
• Understanding of miRNA comes from research on
  siRNA and vice versa
• Some use terms interchangeably

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A Brief History of RNAi
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An Unexpected Result

• Late 1980s, Rich Jorgenson and group
• chalone synthase for deeper purple petunias
• Got white and variegated
• Co-suppression both endogenous and introduced
  genes silenced Napoli et al., 1990
• PTGS but what is the causative factor?
• Similar effects seen in N. crassa Quelling
  Cogoni et al., 1996

From Gura,2000.
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Later, in the C. elegans...

• Antisense RNA injection method for gene
  inactivation
• 1995 characterization of Par1 by Sue Guo
• Essential for embryo polarity
• Did antisense Par1 RNA injections
• Results in embryonic lethality
• Sense Par1 RNA injections gave same result!
• Remained a mystery...
• The basis for the sense effect is under
  investigation and will not be discussed
  further.. Guo and Kemphues, 1995

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Some Sharp Reasoning

• Fire and Mello, 1998
• Both sense and antisense RNAs sufficient for
  silencing
• Silencing can persist, even though RNA is easily
  degraded
• RNA for silencing often generated using
  bacteriophage RNA polymerases
• Specific, but can also make ectopic transcripts
• Maybe some dsRNA in these preparations?
• Could dsRNA be mediating a new silencing
  mechanism?

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Their Experiment

• C. elegans Unc-22 inactivation
• Null phenotype uncoordinated twitching
• Injected sense, antisense, or both into c.
  elegans gut
• dsRNA was orders of magnitude more effective than
  ssRNA
• Effective even in tiny amounts
• Unc-22 null phenotype also seen in progeny of
  injected worms
• Inactivation was due to degradation of target
  mRNA
• Coined the term RNA interference Fire et al.,
  1998

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siRNA Identified

• 25bp species of dsRNA found in plants with
  co-suppression Hamilton and Baulcombe, 1999
• Not in other plants
• 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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Meanwhile, Back in C. elegans

• Discovery of the first miRNA, lin-4
• Non-coding, 22nt RNA
• Identified in screen for defects in timing of
  larval development
• 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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OVERVIEW
RNAi is found in
Fungi, plants and animals
Essential components of RNAi machinery
are conserved across the species
The First Eukaryotic Commandment You dont
produce double stranded RNA
In mammals the dsRNA produces activation of
Innate Immunity PKR system and antiviral response
Small RNAs could produce interference BUT without
induce the antiviral PKR system
Non-specific destruction of dsRNAs Defense
against mobile genetic elements (viruses and
transposons)
This silence was sequence-specific by binding to
target RNA
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Biogenesis and Mechanism of RNAi
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RNAi Two Phase Process

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

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Initiation
Execution
He and Hannon, 2004
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The Basic mechanism of RNAi
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miRNA Biogenesis

• Transcribed from endogenous gene as pri-miRNA
• Primary miRNA long with multiple hairpins
• Imperfect internal sequence complementarity
• Cleaved by Drosha into pre-miRNA
• Precursor miRNA 70nt imperfect hairpins
• Exported from nucleus
• Cleaved by Dicer into mature miRNA
• 21-25nt
• Symmetric 2nt 3 overhangs, 5 phosphate groups

Novina and Sharp, 2004
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siRNA Biogenesis

• Dicer 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
• Allows persistence of RNAi?

Novina and Sharp, 2004
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RNA-induced silencing complex (RISC)
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From Nature Reviews Web Focus on RNAi
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RNA Induced Silencing Complex (RISC)

• RNAi effector complex
• Critical for target mRNA degredation or
  tranlslation inhibition
• Not well characterized 4 subunits? More?
• Activities associated with RISC
• Helicase
• Endonuclease and exonuclease Slicer (or is it
  Dicer?)
• homology seeking/RNA binding
• Preferentially incorporates one strand of unwound
  RNA Khvorova et al., 2003
• Antisense
• How does it know which is which?

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Applying RNAi to Model Systems
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Why All the Hype?

• Quick way to do loss-of-function studies
• Targeting takes long time, lots of work
• Not all loci amenable to targeting
• Cheap

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RNAi in plants, C. elegans, Drosophila

• Introduction of dsRNA sufficient for RNAi
• In vitro transcription
• Chemical synthesis
• Remarkably straightforward C. elegans
• Feed E.coli expressing dsRNA Timmons and Fire,
  1998
• Soak them in dsRNA Tabara et al., 1998
• Common methods transfection or microinjection of
  dsRNA
• Effect lasts days
• Passed onto daughter cells/progeny

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How About Mammals?

• Application of RNAi to mammalian system promising
  for functional studies
• Evidence of RNAi in mammals was harder to
  establish
• Methods for RNAi not a straightforward

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Non-Specific Silencing via Antiviral Pathway
McManus and Sharp, 2002
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Getting Around the Problem

• Critical observation Elbashir et al., 2001
• Size matters
• siRNA (21-22nt) mediate mammalian RNAi
• Introducing siRNA instead of dsRNA prevents
  non-specific effects
• Application via transient transfection
• Dont see persistent or propagative effect as in
  C. elegans etc.
• No RdRP activity identified
• Chemically synthesized
• In vitro transcription

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Empirical siRNA Design Rules

• 21nt long, with 2nt 3 overhangs
• Avoid introns and UTRs
• Avoid regions gt50 GC content
• Use stringent BLAST to help ensure specificity
• Limitations
• Inability to interact with RISC
• Target inaccessibility (structural constraints?)
• Instability of the siRNA

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Still Not Too Efficient

• Usually need to design several siRNAs to get an
  effective one
• Could use a mixture of siRNAs
• Problems
• Increased possibility of non-specific targeting
• Low effective siRNA concentration
• Dont know which siRNA is most potent

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Rational Design of siRNA

• Arising from research on RISC assembly
• RISC contains one strand of the siRNA duplex
  Martinez et al., 2002
• Needs to be the antisense strand to find right
  target
• Can we direct preferential incorporation of the
  antisense strand into RISC?
• Observation 5 end of an siRNA strand is
  incorporated into RISC most efficiently Schwarz
  et al., 2003

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Rational Design Points
Mittal, 2004
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Stable RNAi in Mammals

• Vector driven methods
• Expression of sense and antisense siRNA
• Stable production of siRNA with 3 overhangs
• Expression of pre-miRNA like RNAs
• RNA that folds into hairpin loops with 3
  overhangs
• Act like pre-miRNA dicer substrates
• Some evidence for induction of interferon
  response? Bridge et al., 2003 Sledz et al.,
  2003
• Could do inducible, time, and tissue specific
  RNAi
• Therapeutic potential
• Effective delivery an issue...

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Target Validation

• Luciferase reporter system in HeLa cells
• Tested 15 predicted targets
• 11 validated
• Long way to go...

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New Frontiers for RNA
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RNA as a Molecular Switch

• New class of RNA can act as a switch specifying
  cell fate
• Small Modulatory RNA smRNA Kuwabara et al.,
  2004
• Discovered in mice
• Conserved in vertebrates
• Interacts with regulatory protein
• Turns transcriptional repressor into activator

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Need a Project?

• New roles for RNA add to our current paradigm for
  gene and protein regulation
• Post transcriptional and transcriptional
• Predictive methods, data management, and user
  tools will have to catch up
• Maybe well need a regulomics or RNA
  informatics specific workshop in the future...

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Some strategies for in vivo RNAi
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Alzheimer disease
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Design of miR30.BACE 1 for cloning in viral
vectors
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Lentiviral vectors in gene therapy for
neurodegenerative diseases
Mir30-BACE 1
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Stereotaxic injections
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