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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-sensense 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 Why “no” effect here for sense and antisense RNA? 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 • Short interfering RNA (siRNA -- 21-23 nt long dsRNA) does not activate the interferon response and works to knock down gene expression 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 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: Filipowicz Filipowicz (2008) Nature From: Grohans & WitoldGrosshans &(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) • H. sapiens (mammal)
– ~130 gene families – >400 known genes – 65 conserved families – 100-120 genes found so far Human genome is highly transcribed, even parts that have no known genes Is the extra RNA for si/miRNAs? How to design an effective siRNA Mittal (2004) Nature Rev. Genetics 5, 355 ...
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