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RNA interference: Little RNAs, Big Impact

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Juan Carlos Gallego-G mez. PECET Sede de Investigaci n Universitaria SIU-UdeA. ... pHIT/G. pSUPER-PURO. HindIII. BglII. pNL-SIN-CMV-GFP. ClaI. XbaI. Mir30-BACE 1 ... – PowerPoint PPT presentation

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Title: RNA interference: Little RNAs, Big Impact


1
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
2
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

3
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

4
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

5
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

6
A Brief History of RNAi
7
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.
8
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

9
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?

10
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

11
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

12
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

13
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
14
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15
Biogenesis and Mechanism of RNAi
16
RNAi Two Phase Process
  • Initiation
  • Generation of mature siRNA or miRNA
  • Execution
  • Silencing of target gene
  • Degradation or inhibition of translation

17
Initiation
Execution
He and Hannon, 2004
18
The Basic mechanism of RNAi
19
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
20
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
21
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22
RNA-induced silencing complex (RISC)
23
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28
From Nature Reviews Web Focus on RNAi
29
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?

30
Applying RNAi to Model Systems
31
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

32
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

33
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

34
Non-Specific Silencing via Antiviral Pathway
McManus and Sharp, 2002
35
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

36
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

37
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

38
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

39
Rational Design Points
Mittal, 2004
40
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...

41
Target Validation
  • Luciferase reporter system in HeLa cells
  • Tested 15 predicted targets
  • 11 validated
  • Long way to go...

42
New Frontiers for RNA
43
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

44
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...

45
Some strategies for in vivo RNAi
46
Alzheimer disease
47
Design of miR30.BACE 1 for cloning in viral
vectors
48
Lentiviral vectors in gene therapy for
neurodegenerative diseases
Mir30-BACE 1
49
Stereotaxic injections
50
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