- a mechanism for gene silencing using double-stranded RNA
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.
Fire and Mello received the prize for discovering a mechanism of suppression of gene activity using double-stranded RNA (dsRNA), a process now termed as RNA interference (RNAi).
Use of Model Organism: Caenorhabditis elegans
In this discovery, Fire and Mello worked extensively using the model organism, Caenorhabditis elegans. C. elegans is a widely experimented organism in the field of genetics and developmental biology due to the following reasons:
1. C. elegans is a eukaryotic organism: Eukaryotic cells have membrane-bound organelles which allow the organism to carry out complex functions as well as multiple functions simultaneously, unlike prokaryotic organisms. Eukaryotic DNA is also complexed into chromosomes, similar to all higher organisms.
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2. C. elegans is a multicellular organism: It goes through a complex developmental process involving critical stages like embryogenesis. Hence, discoveries made by experimenting with C. elegans can be directly applicable to many higher organisms such as humans.
3. About 35% of C. elegans genes have human homologs: Homologs1 are described as genes with similar sequences. It has been proven experimentally that human genes can replace their C. elegans homologs when inserted into the C. elegans genome.
4. C. elegans has a small genome size: The C. elegans genome is about 97 Megabases long as compared to the human genome which is about 3000 Megabases long. The entire C. elegans genome was sequenced in 2002.
5. C. elegans is easy to maintain in the laboratory: C. elegans is commonly grown in Petri dishes and they have a relatively fast life cycle. Embryogenesis is completed 12-14 hours after fertilization by sperm entry.
Research Problem: Puzzling observations in plant experiments
In a cell, DNA is transcribed into small messenger molecules called messenger RNA or mRNA which are then translated by ribosomes into protein products. This pathway was termed by British Nobel Laureate Francis Crick as the "central dogma of molecular biology".
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.
A plausible explanation to this phenomenon remained unknown until Fire and Mello's discovery of RNA interference.
The Critical Experiments
Fire and Mello were conducting experiments studying the regulation of gene expression in C. elegans in the 1990s. They focused on the gene encoding for a muscle protein in C. elegans. The hypothesis was that injecting 'antisense' RNA would cause gene silencing as 'antisense' RNA is able to bind to the mRNA molecule encoding for the muscle protein gene.
1. Injecting 'sense4' RNA into C. elegans: Fire and Mello injected 'sense' RNA into the worms, which is an exact copy of the mRNA of the gene but observed no visible effects.
2. Injecting 'antisense5' RNA into C. elegans: Fire and Mello injected 'antisense' RNA into the worms, which was known to be able to bind to the mRNA of the gene, but again observed no visible effects.
3. Injecting a mixture of both RNAs into C. elegans: When Fire and Mello injected a mixture of 'sense' and 'antisense' RNA into the worms, the worms began to display twitching movements. This phenotype is also observed in worms with a non-functional or partially functional muscle protein gene.
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.
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
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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.