Download Different forms of RNA involved in protein synthesis

Clicker question
The 2006 Nobel Prize in Medicine was given to Andrew Fire and
Craig Mello for their work showing that ____ can regulate
the expression of genes.
1)
2)
3)
4)
5)
6)
DNA
RNA
Protein
Carbohydrates
Lipids
All of the above
Clicker question
The 2006 Nobel Prize in Medicine was given to Andrew Fire and
Craig Mello for their work showing that ____ can regulate
the expression of genes.
1)
2)
3)
4)
5)
6)
DNA
RNA
Protein
Carbohydrates
Lipids
All of the above
Fire and Mello discovered that doublestranded RNA activates biochemical
machinery that degrades those mRNA
molecules that carry a genetic code identical
to that of the double-stranded RNA. When
such mRNA molecules disappear, the
corresponding gene is silenced and no protein
of the encoded type is made.
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/
Different forms of RNA involved in
protein synthesis
From Grosshans & Filipowicz, 2008, Nature 451, 414-416
RNA interference (RNAi)
• Introduction of gene-specific dsRNA* into
a cell, resulting in degradation of
homologous mRNA
• Post-transcriptional gene silencing
mechanism
• Natural mechanism that protects an
organism against viruses that produce
dsRNA
ds = double-stranded RNA or DNA
ss = single-stranded RNA or DNA
Early studies in C. elegans (nematode worms)
• 1995 -- tried to use anti-sense RNA to turn off
expression of a gene
• Injection of anti-sense RNA turned off gene
expression
• Injection of sense strand control also turned off
gene expression
• (?)
Guo & Kemphues, 1995, Cell 81, 611-620
From Fire’s Nobel lecture
Double-stranded RNA was known to be relatively stable both chemically
and enzymatically [e.g., 38]. In addition, dsRNA was a known low level
contaminant in synthetic RNA preparations [e.g., 39]. From my graduate
work with RNA polymerases, I was certainly also very familiar with the
sometimes annoying ability of RNA polymerases to start in vitro at ends
and other fortuitous sites. Thus the concept that double-stranded RNA
might be a component of the injected material was hardly a leap of
logic.
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/fire-lecture.html
From Fire’s Nobel lecture
Arguing strongly against dsRNA as a potential effector was the fact that
native dsRNA would have no free base pairs to interact with matching
molecules in the cell. Thus a rational first guess would have been that
injected dsRNA would have been unable to interact specifically with
cognate sequences and thus rather useless for triggering genetic
interference. A critical review of my research plan coming out of the 1997
worm meeting would certainly have brought this up as a major concern.
One could imagine (in retrospect as well as currently) many different
models and explanations for the phenomena. Some scenarios would have
spawned interesting experimental investigations while others would have
been of only limited interest; I was certainly fortunate that our research
grant was not up for renewal for at least a few months.
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/fire-lecture.html
From Fire’s Nobel lecture
Fire looked carefully at preparations of sense and anti-sense RNA
and found that they contained dsRNA.
What you can see is a very prominent band, a bright spot, where the RNA that we
expected was. This photo was deliberately over-exposed to reveal any other
components that might be present, and one can certainly see additional (minor)
bands and a general ”smear” in addition to the major (expected) bands. After a few
preliminary explorations of the dsRNA hypothesis using this assay with these
impure RNA preparations, I was somewhat encouraged but still be no means
convinced. It was clear that a cleaner preparation of starting material was needed.
To achieve this, SiQun cut out the major bands from this gel, extracted the RNA
and injected the purified sense or antisense RNAs into worms. This produced a
result, albeit negative: almost all of the activity was lost by purification of single
strands, suggesting that the sense and antisense weren’t the material that was
causing the interference.
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/fire-lecture.html
Fire and Mello’s experiments in 1998
• Inject dsRNA (sense plus anti-sense strands) into
C. elegans
• More efficient silencing than injecting sense or
anti-sense strand alone
• Just a few molecules of dsRNA per cell were
sufficient to silence expression of the homologous
gene
Only one of the two
strands, the guide strand,
binds directs gene
silencing. The anti-guide
strand or passenger
strand is degraded during
RISC activation.
RNAi and siRNA
• Can knock down gene activity in Drosophila or
C. elegans by introducing long dsRNA
• Long dsRNA (>30 nt) doesn’t work in most
mammalian cells because it initiates a cellular
interferon* response that leads to apoptosis
(programmed cell death)
• Short interfering RNA (siRNA: 21-23 nt long
dsRNA) does not activate the interferon response
and works to knock down gene expression
Interferons are signaling molecules produced in response to viral infections.
How is this done in practice?
• Chemical synthesis of siRNA is expensive and its
effects are transient
• Use a plasmid to express a hairpin-loop structure,
short hairpin RNA (shRNA). shRNA is processed by
Dicer in the cell to produce siRNA
RNAi knockdown experiments can be used to
study the functions of genes in vivo
Review this
slide after
we have
discussed
the HIV
lifecycle
Stevenson, 2003,
Nature Reviews 3, 851
RS virus: respiratory
syncytial virus
(colds, bronchitis)
MicroRNAs (miRNA)
• 20-23 nucleotides, encoded by specific genes
• Processed from long, single-stranded non-coding
RNA sequences that fold into a hairpin
• Function in repressing mRNA translation or in
mRNA degradation
Example of a miRNA in the nucleus before processing
(from http://en.wikipedia.org/wiki/File:Microrna_secondary_structure.png#file)
• Later steps in common with
siRNA pathway
• Inhibits protein translation
and/or induces cleavage of
mRNA
From: http://en.wikipedia.org/wiki/Image:MiRNA_processing.JPG
Dicer in action
From: Grosshans
Filipowicz
(2008)
Nature
From: Grohans & Witold
Filipowicz&(2008)
Nature
451:
414 451: 414
Endogenous miRNAs
• A. thaliana (plant)
– 20 conserved families
– 90 genes
– 72 genes known to function in plant development
• C. elegans (invertebrate)
– 65 conserved families
– 100-120 genes found so far
• H. sapiens (mammal)
– ~130 gene families
– >400 known genes
Human genome is highly transcribed, even parts that have no known genes
Is the extra RNA for si/miRNAs?
Extra slides
How to design an effective siRNA
Mittal (2004) Nature Rev. Genetics 5, 355
From Wikipedia:
“Extensive efforts in computational biology have been directed toward the design of successful dsRNA reagents that
maximize gene knockdown but minimize "off-target" effects. Off-target effects arise when an introduced RNA has a
base sequence that can pair with and thus reduce the expression of multiple genes at a time. Such problems occur
more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of
H. sapiens, C. elegans, and S. pombe that about 10% of possible siRNAs will have substantial off-target effects.[10] A
multitude of software tools have been developed implementing algorithms for the design of general,[90][91] mammalspecific,[92] and virus-specific[93] siRNAs that are automatically checked for possible cross-reactivity.”