module 10 - rnai (2)1
TRANSCRIPT
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The Belgian Blue:
Beef-breed of cattle producing about 40% moremuscle than other cattle.
Produced through selective-breeding.
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McPherron and Lee. 1997. PNAS 94, 12457-12461
Belgian Blue:A deletion in the myostatin gene eliminates the entire active regionof the molecule and it is non-functional
Piedmontese:The coding sequence for myostatin contains a missense mutation.That is, a point in thesequence encodes for a
Different amino acid. Thismutation likely leads to aComplete or nearlycomplete lossof myostatin function.
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Myostatin deficiency has been reported severaltimes in humans.
The strongest boy in the world
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Myostatin: protein that inhibits muscle growth
Human myostatin consists of two identical subunits, eachconsisting of 109 amino acid residues. Its total molecularweight is 25 kDa.
The protein is made in an inactive form. For it to be activated,
a protease cleaves the N-terminus, to produce an active C-terminal dimer.
Myostatin receptors initiate a cell signaling cascade in themuscle, which includes the activation of transcription factorsthat induce myostatin-specific gene regulation.
Myostatin inhibits myoblasts from differentiating into maturemuscle fibers.
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To determine the biological function of myostatin (aka: GDF-8) in micegene function was disrupted by gene targeting (gene knockout)
widespread increase in skeletal muscle mass.
Individual muscles of mutant animals weighed 2-3 times more thanthose of wild-type animals
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Myostatin gene is conserved across animals:
A 2007 NIH study in PLOS Genetics found a significant relationship in
whippets between a myostatin mutation and racing performance.
Whippets heterozygous for a 2base pair deletion in the
myostatin gene were significantlyover-represented in the topracing classes.
The mutation resulted in a
truncated, inactive form of themyostatin protein.
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Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, et al. (2007) A Mutation in the Myostatin GeneIncreases Muscle Mass and Enhances Racing Performance in Heterozygote Dogs. PLoS Genet 3(5): e79.
doi:10.1371/journal.pgen.0030079
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Figure 2. A 2-bp Deletion at Nucleotides 939 and 940 of the Canine MSTN Coding Sequence
Mosher DS, Quignon P, Bustamante CD, Sutter NB, et al. (2007) A Mutation in the Myostatin Gene Increases Muscle Mass and
Enhances Racing Performance in Heterozygote Dogs. PLoS Genet 3(5): e79. doi:10.1371/journal.pgen.0030079
http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030079
http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030079http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030079 -
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The role of myostatin in promoting muscle growth may beused to treat muscle disorders such as muscular dystrophy.
HOW?
introduce substances that block myostatin action
2002, researchers at the University of Pennsylvaniashowed that monoclonal antibody specific to myostatinimproves the condition of mice with muscular dystrophy.
2009, similar results in monkeys.
Other ways such as RNA interference (RNAi)?
inhibit expression of myostatin
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What is RNA interference (RNAi)?
RNAi is a mechanism for regulating gene expression bydegrading mRNAs.
How was it discovered?
How does it work?
The production of an RNA that is complementary to themRNA can repress gene expression.
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3) Slide show:
http://www.hhmi.org/biointeractive/rna/rna_interference/14.html
Online resource and animations:
1) General understanding of mechanism (fun)
http://www.pbs.org/wgbh/nova/body/rnai.html
2) Animation (history and mechanism)http://www.nature.com/nrg/multimedia/rnai/animation/index.html
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RNAi
Many organisms mount RNA interference
response when exposed to foreign genetic
material
The RNAi response leads to degradation of
foreign nucleic acid, thereby protecting the
cell from invasion.
Endogenous developmental processes may
also utilize RNAi mechanism to control gene
expression
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RNA interference.
wildtype extra copies of chalcone synthase gene
show repression of phenotype
First observation: Jorgensen and his colleagues (1980s)
studying petunia.
Added an extra copy of the chalcone synthase gene togenerate more pigment and darker flowers, but observed
the opposite effect:
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A similar observation was made in C. elegans.
In 1995 Guo and Kemphues found that the injection ofantisense RNA to the gene par-1 into the worm blocked par-1 expression
(antisense is complementary to the mRNA and can
potentially bind to it).
Sense strand DNA (used as a control) had the same effect!
Why?
Unlikely to be a simple process of gene silencing by basepairing
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In 1998, Andrew Fire and Craig Mello injected worms with senseRNA, antisense RNA and a mixture of both.
The mixture had a greater silencing effect than either of thesingle strands alone.
They termed this phenomenon RNA interference (RNAi).
What is happening?
dsRNA is cut (DICER)to produce smalldsRNA fragments(siRNAs) that act assilencers ofcomplementarygene sequences.
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How?
RNAi is a mechanism for regulating gene expression bydegrading mRNAs.
RISC binds to siRNAsand to complementaryRNAs.
Target RNAs are cut anddegraded.
Gene expression is
silenced post-transcriptionally throughthe elimination ofmRNAs.
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Argonaute 2 = endonuclease of RISCcomplex that cleaves mRNA
Chu and Rana. 2007.
exogenous
anti-sense = guide strand (target silencing)sense = passenger strand
siRISC
perfect pairing with mRNA
cleaved mRNA cannot be translated.
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siRNAs, miRNAs and piRNAs
short interfering RNAs and Micro RNAs are short ~22
nucleotide single-stranded RNAs
Piwi-interacting RNAs are somewhat longer than siRNAs and
miRNAs
miRNA inhibit translation
siRNA degrade mRNA
piRNAs interact with piwi proteins (related to Argonaute) and
inhibit expression of transposons
Human genome contains ~500 miRNAs and they may regulate
expression of as much as one-third of all genes
How could few miRNAs regulate so many genes?
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miRNAs and siRNAs
miRNA (translational inhibition) siRNA (RNA cleavage)
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miRNAs
MCB, 7thed., Lodish et al., Figure 8-26
First discovered in C. elegansin genetic screens for mutations that
affect development
(lin-4and let-7)
lin-4(22 nt long) and let-7 (21 nt long)
miRNAs hybridize to 3untranslated
regions of targetgenes and inhibit
protein synthesis
STOP AA...
l in-14
STOP AA...
l in-28lin-4
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RNAi effect is not localized
Nature 2004, 431: 338-342
RNAi response has been found to spread to cells at distance
that were not exposed to dsRNA
Systemic response
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RNAi effect requires import and
export of silencing signals
Channel-like proteinsfacilitate transport of
RNA to other cells
Promoter
is specificto
pharynx
PNAS 2007, Winston et al.PNAS 2009, Jose et al.
GFP
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Regulation of X-chromosomeinactivation in mammals
Distinct mechanisms for Counting Choice Silencing
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Recall the earlier lecture in which we discussed the role ofXist RNA in X-chromosome inactivation
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Regulation of X-chromosomeinactivation in mammals
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Competence factor
Xist complex thatinactivates X-chr
Blocking factor
dsRNA that inhibitsXist function
Lee & Lu, Cell, Vol. 99, 4757, 1999
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Regulation of X-chromosomeinactivation in mammals
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Distinct mechanisms for Counting Choice Silencing
Ogawa et al., Science 2008, vol. 320
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Applications of short RNA-mediated gene inhibition:
Research
the ability to eliminate gene function and study the effects
Clinical?
The ability to treat disease and genetics disorders
Eliminate the function of an overexpressed gene that is causinga defect, e.g., overexpression of oncogenes in cancer
Eliminate the function of inhibitors of cell development to treatdegenerative disorders e.g. muscular atrophies and neuraldegenerative diseases.