Hi, in this video, we're going to be talking about RNA interference. So, RNA interference, it can be shortened to RNAi, it's a technique that scientists can use, and they do, to shut off a gene, inactivating it. This is particularly useful if you want to know the function of a gene, knock it out, or stop it, or inactivate it, potentially through RNAi, and then observe what happens when the cell doesn't have that gene anymore. Does it change the phenotype? Does it change metabolism? Does the cell die? Does it grow to huge sizes? Does something change with the cytoskeleton? Any of these types of phenotypes that you look at, you would say, okay. Well, obviously, this gene has a role in cell growth or cell survival or the cytoskeleton or other cellular functions. So, knocking out the gene helps quickly and easily identify the function of that gene. RNA interference is a technique that allows scientists to achieve this knock-out.
How do they do it? Well, they use non-coding RNA, so these are RNAs that don't encode proteins, they remain as RNAs, and that's their function. Examples of these are microRNAs, siRNAs, and shRNA, each acting differently in their ways of how they inactivate a gene. Scientists insert them into a cell or organism in various ways, depending on the type of cells, the organism used, and the RNA used. Different techniques are specific for each cell organism or RNA, but they are put into cells, and these RNA sequences are complementary to some kind of DNA sequence. This is the DNA and the non-coding RNA. What happens is that they bind to the DNA to which they are complementary. These nucleotides tend to bind to similar DNA sequences, and when they bind to it, this recruits other proteins to degrade it, or it'll block the expression of that gene. When that gene cannot be expressed — when it's degraded, when it's inhibited — it's prevented from being either transcribed or translated into a protein, and that function is inhibited. So, RNA interference means putting that RNA into cells, it's complementary to a DNA sequence, and that's going to block the expression of that gene. When that gene is inhibited, as I said before, scientists can figure out the gene's function.
And so, you can do this as I talked about earlier, where you determine the phenotype. So when you knock out the gene, if the cell grows twice its size, then that gene is responsible for controlling cell size. Things like that allow you to determine the phenotype when the gene is not there, but it also allows for confirmation of that. This process is called recovery, or essentially it's replacing that gene. If you knock out the gene and the cell grows to twice its size, then you should be able to put the gene back in and recover the phenotype, and the cell returns to its normal size. These types of experiments are things that scientists are doing all the time using RNA interference to identify gene functions.
Let's look at the example. This is an example of how RNAs are processed. You may have already seen this before if you’ve seen some videos or talked about it in class, over how microRNAs and siRNA generally work. But essentially, in the cell, you start with a double-stranded RNA, an enzyme comes in and cleaves it, it's loaded into another complex, it is still double-stranded, and then that gets degraded. You end up with a single RNA strand bound to the protein it needs to be bound to. Then here is the important part, the important part that we talked about. We have a target sequence here. It comes in. It’s binding. In this case, it’s binding to the mRNA. Some RNAs bind to other RNAs; some bind to DNA, just depending on how they work. In this case, it’s mRNA, and it’s binding that complementary sequence. Right? And when that complementary sequence binds, it's going to target this sequence for degradation, and therefore, that gene will not be expressed, allowing scientists to figure out whatever function that that gene would have normally. So with that, let's move on.
