What can I learn from worms?June 2012
Lesson 6: How do DNA and protein determine behavior?
RNAi: A technique to determine if a protein influences behavior
When you hear the term “RNA” what comes to mind? You might be reminded of DNA and how it is involved in the transcription and translation of DNA to make proteins. RNA serves as an intermediate step between DNA and protein, allowing the information contained in DNA to be translated into a “language” that can be “read” by ribosomes that make proteins. As early as 1939, scientists suspected that RNA played a key role in protein synthesis. However, it was not until 1965 when Robert W. Holley discovered the sequence of yeast tRNA (transfer RNA), the type of RNA responsible for reading mRNA (messenger RNA) and translating that message to a corresponding amino acid code that produces a protein. Without RNA, the cell could not survive! It is an essential molecule that every organism, from planarian to human, must have in order to produce proteins and sustain life.
While RNA is an essential step in creating proteins, it has also been found to protect the cell against foreign invaders such as bacteria and viruses. Certain types of viruses contain double-stranded RNA (dsRNA) and over thousands of years, cells developed a method to degrade, or cut, dsRNA so that it can be disposed of. In 2006, two scientists, Andrew Fire and Craig Mello, received a Nobel Prize Award for taking this idea of dsRNA a step further for research purposes. They found a sense RNA sequence of a particular muscle protein in the worm C. elegans. With this code, they created an anti-sense single strand RNA(ssRNA) and then injected both the sense and anti-sense RNA strands into C. elegans, simulating a viral dsRNA sequence. Once injected, dsRNA started a chain reaction, where not only the injected dsRNA molecules were degraded but also other RNA molecules that were identical to the sense and antisense RNA molecules of the particular muscle protein. Fire and Mello noted that the C. elegans injected with dsRNA moved similarly to worms who had a defective gene-indicating that the muscle protein had decreased significantly! An alarm had sounded in the C. elegans’ cells when the dsRNA had been injected and formed dsRNA. Thus, the researchers had created a way to easily “knock down” or decrease the amount of protein in an organism’s cells by simply injecting the anti-sense RNA of a known RNA sequence that codes for a specific protein.
This technique created by Fire and Mello was thus named RNA interference (RNAi). While this technique is often used as a way to determine the function of a protein, RNAi experiments actually examine how a system or organism responds or adapts to the loss of this protein. While there are many proteins involved in RNAi, this article will focus on the major players in this process. To begin, when dsRNA is detected, an enzyme called ribonuclease gets activated and will cleave, or cut, the dsRNA into smaller pieces. These small dsRNA pieces then get dissociated into single strand RNAs (called short interfering RNAs – or siRNA) and become integrated into a complex called RNA-induced silencing complex (RISC). These RISCs can move around the cell looking for any RNA strands that match the sequence of the siRNA within the RISC. This step is crucial-as noted in the Fire and Mello experiment in Figure 2. Without the dsRNA, the RISC will not get activated; it must have the dsRNA precursor to form the siRNA. That is why the anti-sense RNA alone did not cause the mutant muscle movement in C. elegans.
A scientist can introduce RISC with a specific siRNA into multicellular organisms such as a planarian or a mouse. As a result, the RISC will destroy both the siRNA molecules that were introduced by the scientist AND any endogenous mRNAs that match the siRNA sequence. For example, if a researcher wants to knock down a protein thought to help a planarian sense its food, the researcher could find the sequence of the gene and then create a single strand RNA copy of the gene. With this siRNA, he/she can integrate the siRNA into RISC, insert this complex into the planarian and then monitor the planarian’s behavior with the protein of interest knocked down.
RNAi has been a useful technique for knocking out or knocking down proteins in adult organisms. A protein of interest can be diminished or knocked down in adult animals, without the risk of changing the protein’s function during early development. That is, the function of the protein can be studied in a wild type, non-mutated, adult animal.
It is important to note that when studying knockout animals, you are studying how the organism (cells, tissues, proteins, etc) adapts to maintain normal function when the targeted protein is missing. Many times there are several proteins that can perform the same function and can compensate when one protein is knocked out.
Using the image and text above, draw a flow chart to represent how RNAi can be used to study planarian protein function.
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