Silencing RNAs in Mammals

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Silencing RNAs in Mammals Michael T.
McManus MIT Center for Cancer Research
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what is RNA interference?
RNAi is a way to silence gene expression
to perform RNAi, dsRNA homologous to the
targeted gene is made and then introduced into
cells
nucleus
any mRNA with high sequence homology to the
dsRNA may be silenced
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RNAi a tool for inhibiting gene expression in
vivo

• C. elegans (Fire et al., 1998)
• Drosophila (Carthew et al., 1998)
• Planaria (Newmark et al., 1998)
• Trypanosomes (Ullu et al., 1998)
• Hydra (Lohmann et al., 1999)
• Zebrafish (Wargelius et al., 1999)
• Mice (Wianny Zernicka-Goetz, 2000)
• cosuppression in plants
• quelling in Neurospora

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practical aspects of RNAi

• biological research
• defining gene function (gene knockout)
• C. elegans genome RNAi projects
• defining biochemical pathways
• microarray screening of RNAi knockouts
• therapeutic treatment
• cancer
• viral infection
• parasitic infection

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How does RNAi work?
RNAi works postranscriptionally.. in key two
steps!
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step one
processing the dsRNA into 21-23 nt fragments
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Dicer contains two RNAse III domains
long dsRNA

siRNAs
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siRNAs have a defined structure
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step two
the antisense strand of the siRNA guides cleavage
Tuschl, Cell 2002
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RNAi silencing complex

• may be associated with translating ribosomes

• active RNAse enzyme not yet identified

• may participate in endogenous pathways that
  silence genes via translational repression

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Model for RNAi
siRNA
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Initial basic questions about mammalian RNAi
how do you get siRNAs into cells?
how long does it last?
can any region of a gene be effectively targeted?
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how to get siRNAs into the T-cells
cationic lipids, calcium phosphate, etc.
dead cells
T-cell
receptor-dependent transport, endocytosis, etc.
no silencing
electroporation
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develop an assay quantitative on the single-cell
level
T-cell
CD8
flow cytometry detector
CD4
fluorescent antibodies detect expression on the
single cell level
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transfection of plasmids and siRNAs
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can any region of the mRNA be targeted with
siRNAs?


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Influenza mRNA target-sites
Ge et al PNAS, 2003
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how long does the RNAi response last?
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miniconclusion
RNAi works by target degradation of the mRNA
RNAi creates knock-downs, not knockouts!
not every siRNA works
RNAi via siRNAs is transient, lasting 3-6 cell
doublings
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establishing long-term RNAi let the cell make
the siRNA for you!
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Hairpin RNAs
RNAi silencing hairpins microRNAs
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hairpin siRNAs
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CD8 hairpin RNAs
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establishing long-term RNAi
Let the cell make the siRNA for you!
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(No Transcript)
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lentiviral construct for siRNAs
Rubinson et al Nature Genetics, 2003
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Lentiviral CD8 knockdown
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stable 14-fold CD8 knockdown by lentivirus siRNAs
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functional silencing of genes in ES cell-derived
mice by lentivirus-induced RNAi
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Efficient generation of transgenic mice through
zygotic lentiviral infection
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overview
McManus and Conklin RNAi, 2003
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mini-conclusion
Although silencing by siRNAs is transient,
vectors can be made to express siRNAs in cells
RNAi knock-down mice can be generated in lt30 days
Hairpin-RNAi mice do not appear to have gross
developmental defects (interference with the
endogenous hairpin RNA pathways)
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acknowledgements
CD8 lentiviral studies Chris Dillon Doug
Rubinson Adam V. Kwiatkowski Claudia Sievers Lili
Yang Johnny Kopinja Frank B. Gertler Martin L.
Scott Luk Van Parijs
for protocols, vectors etc http//web.mit.edu/cc
rhq/vanparijs/
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two Dicer-related pathways
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small temporal RNAs (stRNAs)
Ambros lab, Rukvun lab
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extensive class of RNAs
Lagos-Quintana et al Science, 2001
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conservation of microRNAs
miR-1
Lau et al 2001
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miRNAs can be grouped into families
Tuschl, Ambros, Bartel labs
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Major questions in the miRNA field
What do the miRNAs do? what genes do they target?
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predicting targets of microRNAs
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anatomy of a miRNAmRNA interaction
nonpaired 5/3 ends
mRNA bulges
3-UTR
A
GUU
AUU
5-UUAUACAACC CUAC CUCAACGCGAUGUAAAUAUCGCAAUCCCUU
UUUAUACAACCAA CUGCCUC-3


3-GAUAUGUUGG GAUG GAGU-5
3-GAUAUGUUGG GAUGGAGU-5
miRNAs
AU
AU
miRNA bulges
GU wobble
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McManus and Sharp NRG 2002
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search strategy
Create a loose pattern that resembles a
let-7lin-41 miRNA target site
rank UTR hit list to refine candidates
Search a database for UTRs that contain the
pattern(s)
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let-7 stRNA against Ce UTR dataset
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let-7 predicted targets in C. elegans
67 predicted targets that contain at least two
let-7 sites
at a first glance 31 developmental genes 6 are
known to be heterochronic 5 have vulval
functions 5 are nuclear hormone receptors 3 are
related to neuronal functions
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lin-4 randomization
number of hits
sum of deltaG
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conclusions
RNAi knock-down mice can be generated in lt30 days
and vectors are freely available for the
scientific community
mRNA targets of microRNAs have been predicted and
are being tested
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acknowledgements
miRNA target prediction Aadel Chaudhuri-UROP John
Obenauer-Yaffe Lab Jalagani Srineel Michael Zuker
Phillip Sharp
MIT Center for Cancer Research Cancer Research
Institute
http//web.mit.edu/mmcmanus/www/RNAi.html
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acknowledgements
miRNA target prediction Aadel Chaudhuri-UROP John
Obenauer-Yaffe Lab Jalagani Srineel Michael Zuker
STOP! Do not go beyond THIS slide! Doing so may
bring down dppt!!!!
Phillip Sharp
MIT Center for Cancer Research Cancer Research
Institute
http//web.mit.edu/mmcmanus/www/RNAi.html
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acknowledgements
STOP! Definitely do not go beyond THIS
slide! Doing so WILL bring down dppt!!!!
miRNA target prediction Aadel Chaudhuri-UROP John
Obenauer-Yaffe Lab Jalagani Srineel Michael Zuker
Phillip Sharp
MIT Center for Cancer Research Cancer Research
Institute
http//web.mit.edu/mmcmanus/www/RNAi.html