RNA Interference. By Matthew Sach, Clara Summers, Bryan Solomon. An Introduction…. In between genes, there are long stretches of DNA that researchers originally thought were not functional, or ‘junk’.
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RNA Interference By Matthew Sach, Clara Summers, Bryan Solomon.
An Introduction… • In between genes, there are long stretches of DNA that researchers originally thought were not functional, or ‘junk’. • However, recently it has been discovered that some stretches of this DNA (up to 30%) are firewalls against viruses and ‘jumping genes’, or transposons, which damage cell DNA. • The RNA that this DNA transcribes to ‘interfere’ in the expression of genes by destroying or halting the translation of mRNA, and thus the process has been dubbed RNA interference, or RNAi for short.
What does RNAi do? • RNAi is found in many eukaryotes and is used to moderate gene expression. • In most cases, it causes a gene to be silenced, or not expressed. • In some cases, however, it can affect an inhibiting gene for a certain protein, and thus act as an up-regulator.
Some History • RNAi was first observed in plants, specifically petunias, but was not recognized as such. • Researchers into plant genetics wished to increase the pigmentation in a species of purple petunia by adding more RNA of the same genetic sequence that translated into the specific purple pigment. • However, instead of creating a more vividly colored flower, the researchers were instead presented with variegated petals.
C. elegans • Two ‘worm guys’, Craig C. Mello and Andrew Z. Fire, came across this interesting result and were intrigued. • They decided to test this using C. elegans, targeting a muscle protein as this could be easily observed and tested. • After attempting to suppress production of this protein using mRNA and antisense (backwards) RNA, they finally found that using double-stranded RNA was successful, and thus discovered the mechanism of RNA interference.
The Science • The stretches of DNA that code for RNAi fragments contain two complementary strands of DNA separated by a section of uninteresting DNA. • When this stretch is translated into RNA, it folds back on itself, forming a hairpin shape. • This hairpin is called a pre- micro-RNA (pre-miRNA). • The loop is then cleaved from the rest of the pre-miRNA to form a double-stranded RNA (dsRNA)
The Science Part II • The dsRNA is cut up into small interfering RNAs (siRNA) by a protein called Dicer. • These siRNA then seek out RNA-induced silencing complexes (RISC) that contain endoribonucleases, i.e. enzymes that chop up RNA. • The siRNA then bond to messenger RNA (mRNA) with the same sequence. • This bonding activates the RISC, which then cleaves the mRNA, thus destroying its function. http://www.ambion.com/techlib/append/RNAi_mechanism.html
The Science Part III • Alternatively, if, when the hairpin loop is removed, the pre-miRNA still has non-complementary strands, it is considered miRNA. • These miRNA again combine with the RISC complexes, and attach to mRNA. • However, instead of signaling the mRNA for destruction, they inhibit translation.
In the Lab. • RNAi has been found in many eukaryotes, although not all, making it worth investigating. • RNAi has quickly become a very useful tool for investigations into genetics and protein function. • There are currently two methods for using RNAi in the lab; inserting dsRNA, or using siRNA • Both methods utilize the inherent ability of most eukaryotes to process RNAi fragments.
dsRNA vs. siRNA • siRNA is however, easily absorbed into the RNAi pathway in any cell. • However, it is much harder to keep viable as it is less stable due to its small size. • dsRNA is useful because it is more stable. • However, it also invokes a strong intra-cellular immune response in higher eukaryotes, as it is rare and thus is interpreted as a viral attack. • This makes it useful for investigating the effects of RNAi in, for example, C. elegans or Drosophila, but less useful in mammalian cell cultures.
How do we get the RNA in there? • Inserting dsRNA and siRNA into a cell or organism is a very costly and time-consuming process. • When working with most organisms, the best way is to soak the cells in a solution of the desired RNA and hope it gets picked up by the cell. • However, in C. elegans, scientists have found that feeding the worm bacteria rich in the right RNA will cause the worm to pick up the RNA. • The mechanism for this is unknown, but has seriously reduced the cost of this procedure. • Alternatively, researchers at Yale University have recently discovered a method of transporting siRNA using FDA approved nanoparticles. • It is hoped that this might eventually find a use in clinical treatment of medical conditions that could be relieved by RNAi.
Uses • RNAi has many uses in the fields of genetics, medicine, and biotechnology. • For example, it could potentially be used to treat human genetic disorders, such as macular eye degeneration. • Also, by disrupting gene expression through RNAi, researchers can discover the function of specific genes and groups of genes.
Other Uses • It is currently being investigated whether RNAi has a use in contraception, by preventing production of the protein that binds sperm to an egg. • This has been tested in mice, but is not yet in human trials. • Also, RNAi has found a new home in the field of genetically engineered foods. • Researchers hope to drastically reduce the amount of the amusingly named gollypol, a toxin, in cotton, so that it can be used as food source high in dietary protein. This research also applies to the cassava plant.
Animation • http://www.nature.com/focus/rnai/animations/animation/animation.htm
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