- Introduction
In the early 1990s, scientists did a surprising discovery in petunia plants. While trying to deepen the purple color of their flowers they tried to overexpress the pigment-producing gene under the control of a powerful promoter. However, instead of the expected deep purple color, many flowers appeared lighter or even white, a process called post-transcriptional gene silencing, or PTGS. A breakthrough discovery came in 1998 when scientists discovered that this phenomenon was not limited to plants, but also found in higher organisms like the nematode Caenorhabditis elegans. Even more important, they found that the mediating factor was double stranded RNA (dsRNA). Therefore, this process was denoted as RNA interference or RNAi. Scientists quickly realized the potential of RNAi as a new and powerful tool in loss-of-function studies.
- RISCy business
In recent years a lot of effort has been put in unravelling the working mechanism of RNAi. The process starts with the cleavage of input dsRNA into 21-23 basepair small interfering RNAs (siRNAs) by the enzyme Dicer in a processive and ATP-dependent manner (see figure). Successive cleavage events degrade the RNA to 19-21 bp duplexes, each with 2-nucleotide 3' overhangs. Next, the siRNA duplexes bind to a nuclease complex to form what is known as the RNA-induced silencing complex, or RISC. Unwinding of the siRNA duplex is required for activation of the RISC complex, which then targets the homologous messenger RNA (mRNA) by base pairing interactions and cleaves the transcript, resulting in efficient gene silencing.- Short Hairpin RNAs
RNAi mediated gene silencing is usually achieved by administration of synthetic oligonucleotides. There are two big disadvantages with this method however. First, it is very expensive to produce these oligos. Second, the effect lasts only a few days after transfection. These two problems were overcome by the development of plasmid-based short hairpin RNA systems, like pSUPER. The idea is to generate a DNA encoded short hairpin construct, driven by a strong PolIII promoter, which in cells is transcribed into a short hairpin RNA. The hairpin is subsequently processed to form a functional siRNA duplex (see figure). The hairpin containing vectors are relatively easy and cheap to produce, and can be stably transfected into cell for long-term loss-of-function studies.- Rules, Rules, Rules
Until recently, selecting the proper target sequence within a gene was largely based on trial-and-error and multiplicity. In 2003 however, two Cell papers claimed to have discovered the "golden rule" for RNAi target selection. Work from Khvorova et al. and Schwartz et al. showed that thermodynamic properties underly the selection of the proper RNA strand for loading into the RISC complex. In short, if the 5' anti-sense end of the duplex basepairs relatively weak in comparison to the 3' anti-sense end, the proper (anti-sense) strand is loaded, resulting in efficient knock-down of the gene (see figure). If the reverse is true, the wrong (sense) strand is loaded, and silencing is highly inefficient. These papers suggest that relatively simple calculations of thermodynamic parameters of potential target sequences would be sufficient to predict silencing efficiency. As a result a number of papers were published all kind of scoring algorithms and with even more rules. Unfortunately, all this work was performed with synthetic oligonucleotides, and it remains to be seen if these rules apply for plasmid-based RNAi systems like pSUPER as well.
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RNAi is thought to be the immune system of our genome, protecting it against insertions of viral DNA and transposons. In a sense it can be compared to role that restriction enzymes play in bacteria.
In nematodes like C. elegans RNAi spreads systemically from cell to cell. Consequently, one can simply administer the double stranded RNAs by simple injection or through food uptake. It is even possible to feed the worm bacteria that contain plasmids encoding the RNAi duplexes. The bacteria express the duplexes, which are taken up via the intestines of the worm, and spread systemically throughout the animal.
Transposons are segments of DNA that can move around to different positions in the genome of a single cell, thereby causing mutations and changes in genome size. The so-called retro-transposons move by a copy-paste mechanism using an RNA intermediate. Their exact function is still a debate, but it has been estimated that about 40% of our genome consist of retro-transposons. They have been called "junk" DNA and "selfish" DNA. "selfish" because their only function seems to make more copies of themselves and "junk" because there is no obvious benefit to their host.