In 2006, the Nobel Prize in Physiology or Medicine was awarded to two American scientists, Andrew Z. Fire and Craig C. Mello. Fire is currently a Professor of Pathology and Genetics at the Stanford University School of Medicine, Stanford, CA, USA and Mello is a Professor of Molecular Medicine at the University of Massachusetts Medical School, Worcester, MA, USA.
Around 1990, scientists working with red petunia flowers were trying to amplify the redness of the petunias. They hypothesized that by inserting a gene coding for the red pigment into the flowers, the red hue can be intensified, hence beautifying the flower. However, unexpected results were observed - the flowers produced no pigment at all and became white. At a molecular level, it was discovered that the mRNA coding for the red pigment had "disappeared". This indicated that gene silencing occurred at either a transcriptional2 or post-transcriptional3 level.
Together, 'sense' and 'antisense' RNA form double-stranded RNA. From the experiment, it can be assumed that this dsRNA molecule was responsible in inactivating the mRNA molecule encoding for the muscle protein gene. However, since dsRNA molecules have no free sequences that can bind to mRNA molecules, it was assumed that the gene silencing occurred by a yet unknown mechanism.
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In another experiment with another C. elegans gene, Fire and Mello investigated for the same effect in C. elegans embryos. They injected 'antisense' RNA and a mixture of 'sense' and 'antisense' RNA to C. elegans embryos. By using a special staining technique, they could observe the mRNA activity of the embryos post-injection.
The results were similar as their earlier experiment.
1. Uninjected embryo: Intense staining was observed. This was the benchmark, positive control for mRNA activity.
2. Embryo injected with only 'antisense' RNA: Slightly less staining was observed. Conclusion was that mRNA activity was hindered by some degree.
3. Embryo injected with both RNAs: No staining was observed. Conclusion was the mRNA activity was completely gone and hence, the gene had been silenced.
After many other experiments involving C. elegans genes, Fire and Mello made the following conclusions in their article in Nature:
Gene silencing was triggered effectively when dsRNA was injected into the organism but weak or no result when ssRNA was injected.
When dsRNA was injected, the mRNA disappeared - degraded and broken down.
The injected dsRNA sequence must match the final version of the mRNA, which is after splicing out the intron sequences. Hence, this suggests RNA interference occurs post-transcription.
Only the mRNA that corresponds to the injected sense RNA of the dsRNA was silenced. Hence, this suggests that RNA interference using dsRNA is specific for each gene targeted.
RNA interference is a catalytic process - only a few dsRNA molecules were needed to elicit the gene silencing process effectively, suggesting that enzymes were involved.
The effect of the dsRNA could spread between tissues and even down to the next generation.
Their findings were phenomenal in explaining many puzzling and unexpected experimental results and uncovered a natural mechanism for regulating the flow of genetic information.
The RNAi Machinery: A Molecular Mechanism
In the years following Fire and Mello's discovery of RNAi, the different components comprising the RNAi machinery were discovered. When dsRNA enters a cell, it binds to a large protein complex called Dicer which cleaves the dsRNA molecule into small fragments. These fragments bind to another protein complex called RISC (RNA-induced silencing complex). The 'sense' RNA strand is eliminated while the 'antisense' strand serves as a probe6 that can bind to mRNA molecules. When the corresponding mRNA is found and bounded to the RISC complex, it is cleaved and degraded similar to how endonucleases7 cleave DNA.
Significances of the Discovery of RNAi in the Fields of
Developmental Biology and Genetics
1. RNAi in the development of organisms: Shortly after the discovery of RNAi by Fire and Mello, a class of several hundred genes in the human genome that encode for small RNA molecules termed microRNA were discovered. microRNA genes are translated into hairpin-like structures of dsRNA molecules which are fragmented by the Dicer complex into RNA fragments which then bind to the RISC complex which bind to mRNA molecules and degrades them, blocking protein synthesis. This system of regulation of gene expression has been seen to be essential for eukaryotes - single-cell organisms to humans. RNAi plays a significant role in turning off genes during the development of an organism.
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2. A new experimental technique to silence genes specifically: The discovery of RNAi led to an immediate suggestion that this phenomenon can be used as an experimental technique in reverse genetics8. By suppressing specific genes to look for the subsequent phenotypic effects, it could be possible to map out the function of virtually any gene in an organism.
3. A possible tool used in gene therapy of the future: RNAi can be used to silence genes that cause many human diseases where genes are usually overly expressed.
The discovery of RNA interference for silencing genes in eukaryotic cells by the recognition and processing of dsRNA molecules was astounding and has rapidly led to many other discoveries in the field of developmental biology, genetics and medicine. Amazingly, the RNAi mechanism is able to process both exogenous and endogenous dsRNA. Development of an organism relies on RNAi to appropriately silence genes at the appropriate time to ensure the proper function of its cells and tissues. Finally, RNAi has empowered us with an incredible new experimental technique to study gene function by the means of reverse genetics which can lead to the discovery of future applications of RNAi in treating human diseases.
Glossary of Terms
Homologs1 - Genes with a similar sequence.
Gene silencing at the transcriptional level2 - Synthesis of mRNA product is affected.
Gene silencing at the post-transcriptional level3 - Finished mRNA product is affected.
Sense4 - A coding strand of DNA or RNA.
Antisense5 - A non-coding strand of DNA or RNA. Antisense DNA strands serve as the template DNA to synthesize mRNA molecules. Antisense RNA molecules can be used to bind and silence mRNA molecules.
Probe6 - A probe is a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome.
Endonuclease7 - An enzyme that degrades DNA or RNA by breaking it into smaller fragments
Reverse genetics8 - Finding out the function of a specific gene by silencing it as opposed to forward genetics9.
Forward genetics9 - Finding out the specific gene that results in a certain phenotype.