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Lecture 24

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

nd BIOL 300 November 2 2012 Lecture 24 Dr. Shock The work that led to the Nobel prize related to small RNAs was based on knocking out genes through injection of double stranded RNA molecules (which is commonly done now) • It took years of research to realize that this mechanism is actually the same mechanism as the lin4 mechanism This all began with the idea that you could knock out gene expression by introducing antisense RNA • The idea was that the antisense RNA would bind and inactivate the mRNA by preventing translation They observed that: • You would get a mild phenotype when you injected antisense RNA • However, when you injected sense RNA (same sequence as the gene), you would get no phenotype (i.e. same as wild-type) • When you injected double stranded RNA (both an antisense and a sense sequence), then you would observe a strong phenotype • This was done basically randomly (people didn’t even know that double stranded existed at the time) This experiment was done with a gene known as unc-22, for which they already determined a phenotype of twitching based on mutational analysis • Therefore, injection of double stranded RNA would lead to the same phenotype as mutational analysis, whereas only the antisense would be a mild phenotype They also had to carry out the following control mechanisms: • They had to check if it was just the injection of any dsRNA that would make the animal sick; they did this by injecting a random dsRNA that was not complementary to the mRNA, and found no phenotype • You also want to know if you’re really knocking out unc-22 to control the phenotype, or some other gene • They solved this by knocking out unc-22 through dsRNA injection, and also injecting a mutated unc-22 gene to which the dsRNA would not be able to hybridize 1 nd BIOL 300 November 2 2012 Lecture 24 Dr. Shock • Remember, the dsRNA is only complementary to a small region of the target gene • What they observed is that injection of this mutant unc-22 restored the wild-type phenotype, which ensures that the dsRNA is specific to the unc-22 gene to produce the phenotype (i.e. the dsRNA does not bind all kinds of RNA) • This also shows that the complementarity of the dsRNA and the part of the unc-22 gene is required to cause this phenotype Additionally, you could conduct an RNA in-situ hybridization technique to display the amount of gene expression in the cells (you could also do a Northern Blot fo achieve the same results, except the RNAs are result on a gel) • In this case, they used labelled antisense RNA as a probe to see how much mRNA of the unc-22 gene is in the cell • As expected, they found that: • When you inject only sense mRNA and a probe, you see the darkest color to represent the most mRNA (i.e. the highest level of gene expression) • When you inject only antisense mRNA and the probe, you see a lighter color • When you inject the dsRNA and a probe, you would see no color (no gene expression) They then tried to figure out which RNA sequence(s) they needed to observe the phenotype • They injected more dsRNAs with different sequences complementary to different regions of the unc-22 gene (e.g. an intron) • Testing on an intron would determine whether or not the dsRNA works on unspliced RNA • The dsRNA does not work on introns or promoter sequences, suggesting that the mechanism used by dsRNA is post-transcriptional (i.e. after mRNA modifications) They then carried out experiments to see how much of the correct dsRNA you needed to observe the full phenotype • They did this by incrementally injecting less and less dsRNA until they no longer observed a phenotype • It turns out that the dsRNA mechanisms is concentration independent (it works if you inject a lot of dsRNA, but also if you inject very small amounts of dsRNA) • This suggests that the dsRNA works through a enzymatic process (i.e. the dsRNA is not used up in the process) • There must be some enzymes involve which somehow process the dsRNA This is now a widely used mechanism to targeted gene knockouts. A few years later, people began to understand what really happened in the dsRNA mechanism. Where does dsRNA come from? • As an experiment, you could inject it, but there must also be a source of dsRNA naturally occurring in the cell • It turns out an RNA strand like lin4 actually folds over on itselt to form a stem-looped structure with a double-stranded component • Also, there are viral genomes that can be double-stranded (in which case the cell needs to destroy them) 2 nd BIOL 300 November 2 2012 Lecture 24 Dr. Shock Whenever the cell encounters the dsRNA, an enzyme known as Dicer will chop up the dsNRA into small pieces (essentially a defence mechanism for viral genomes)
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