So last of these particular features that we're going to talk about in terms of explaining epigenetic control are noncoding RNAs. And, as the name suggests, they don't code for protein but rather just code for RNAs, the genes just code for RNAs. So there are many classes of these non-coding RNAs, and we're only going to think about three. There are classes that are small, medium in length, or long in length. And the ones that we're going to talk about, we're going to focus on the Piwi-interacting RNAs. And that's because they're able to alter the epigenetic state. And secondarily, the long non-coding RNAs again because they seem to be able to alter epigenetic state by directing the epigenetic machinery. The additional one I'd start thinking about, start by talking about are the MicroRNAs or miRNAs. So, while these don't alter epigenetic state, they're important as a comparison to the Piwi-interacting RNA or the piRNA's. And also they're used very widely, or at least the biogenesis of microRNAs are taken advantage of for experimental systems widely throughout the world. And so it's quite nice to talk about them anyway, even though they don't actually alter epigenetic state. So the micro RNAs are really involved in post-transcriptional gene silencing. That is, they're involved in halting translation or in degrading the messenger RNA so that you'll get less protein product. But they don't go back and alter the epigenetic state of the chromatin. Though first discovered in worms and plants, but they've also been characterised in mammals now. And there are more than 1,000 micro RNA genes in mammals. So these genes are found in the genomic DNA in the nucleus, and when these MicroRNA genes are transcribed, they make a primary microRNA, or a pri-miRNA. In this pri-miRNAs have these stem loop structures, and the stem loop structures as you can see here are recognised by the Drosha enzyme which will then cut off that stem loop and have it exported from the nucleus. When it's exported from the nucleus it will be bound by a second enzyme called Dicer. And once Dicer has processed these three pre-microRNAs now, then we have a miR duplex a MicroRNA duplex. Or indeed a small interfering RNA duplex or an siRNA duplex and these double stranded RNA's are then loaded into the RISC complex, which is a complex of proteins. So this, this is where there can be two outcomes. The outcomes can either be, that if there are some mismatches between the sequence that's found in this MicroRNA, and a messenger RNA, then it can bind with this mismatch as shown here. And this actually leads to, translational repression. So it will decrease the likelihood that the messenger RNA will actually be translated into its protein product. And this is what mostly happens in animal cells. So this is the primary function, the endogenous function of MicroRNAs. Whereas, in plants, what primarily happens is that you get mRNA cleavage. So the microRNA is incorporated into the RISC complex. However, this time, if there's perfect identity, there's a perfect homology between the microRNA and the messenger RNA. And now the target is cleaved, and so if you chop up the messenger RNA, clearly it, the protein can no longer be translated. So this does happen in most cases in plants. As I said, but it's also, if we take advantage of this mechanism of gene silencing, of post-transcriptional gene silencing, in any context. By using siRNAs or small interfering RNAs, or indeed if we design a microRNA to have perfect identity, a perfect match, perfect homology with a particular gene of interest, then it tends to go through this type of mechanism. This miRNA cleavage mechanism. So we know that microRNAs are really important in development and differentiation. And that they really give a very fine level of control of gene expression at the post-transcriptional level. We also know they are misexpressed in various diseases and in cancer. And we'll come back to this when we talk about cancer epigenetics in later lectures. But I think one of the interesting things as I mentioned is that these microRNA. So the microRNA machinery is harnessed in a lab based setting to be able to do experiments and this tends to be called knock down or RNAi, so RNA interference. This is used in many labs around the world including my own. So I will just spend another slide telling you briefly about how this relates to my work. So you'll remember just in the last lecture or two lectures ago. I said that we try to look at about 1000 genes and how these potential epigenetic regulators are involved. Whether they're involved in epigenetic modification of the genome. The way that we actually do this is that we define, we build microRNAs that are targeting each of these thousand genes. So, many many microRNAs that individually target a single one of these factors. These thousand or so potential epigenetic regulators. And this means that we can reduce the expression of each of these in turn. Because these will lead to mRNA cleavage. And will reduce the expression of each factor, one by one. And this really is a powerful mechanism to be able to study the function of these genes. So, what we do is we consider that these, around a thousand or so potential epigenetic modifiers, a bit like a big pile of puzzle pieces that we don't understand anything about or very little about. And now what we want to do is go through it and screen for those that perform or function in areas that we're interested in. So we look at X inactivation, and you'll remember that I mentioned what X inactivation was in earlier lectures. The dosage compensation in female mammals. We look in different systems in mice, so the blood system or in the brain or in indeed embryonic stem cells, so these early stem cells that have, the capacity to form an embryo. And in each of these scenarios what we're interested to do is say ‘Which of these puzzle pieces are really playing a role in each case?’ So it might be that we can, when we've performed these screens, that we will be able to assemble how these puzzle pieces interact with one another in each of these different cellular scenarios, and be able to piece together the mechanism of epigenetic control. And the reason we're able to do this is because we can harness this knowledge about microRNA biogenesis. And use it in the lab to be able to reduce the expression of each gene in turn, in a post-transcriptional way.
