So the next class of non-coding RNA's, we'll think about are the Piwi interacting RNA's or piRNA's and these are one of the much more recently discovered classes of non-coding RNA's. So they're best characterised in Drosophila and probably in worms, otherwise known as C. Elegans. So, but we do now understand a little bit more about how they function in mammals. What they seem to be doing is they play a role in silencing the transposable elements. These are those repetitive elements that we've described, that I've described before. That are able to transpose themselves or jump around the genome. So these particularly involved in silencing the transposable elements, piRNAs are silencing them in the stem cells and in the germ line. And this is to be able to maintain genomic stability. So to remind you what we've spoken about before with transposable elements or repetitive elements was that they can, first of all, they can jump out and place themselves somewhere else in the genome and this causes disruption, genomic disruption. They also have very strong promoters which can act out into the neighbouring genes and disrupt the transcription of neighbouring genes. And of course because they are repeats they're the same as one another. They can cause aberrant recombination between chromosomes that should not otherwise be recombining. But if they can be silenced, epigenetically silenced, these would decrease all of those functions. And limit the damage that piRNA, that the the transposable elements can cause. So the piRNAs are helping to maintain genomic stability in that way. Perhaps the reasons this is more important, or that it happens more often in the germ line and stem cells is because these are the occasions when the genome under goes a lot of epigenetic reprogramming and many epigenetic marks are removed from the genome. And therefore, the transposable elements, the previous epigenetic silencing needs to be reestablished, and so we think the piRNAs are involved in this mechanism. So, they're longer than the microRNAs. These ones are 24 to 35 nucleotides rather than being the shorter 19 to 24 as for the microRNAs. They are transcribed from the transposons themselves. And they either have uni-directional promotors or bi-directional. they're also transposed not just from complete transposons, but from transposons or repetitive element remnants that are found throughout the genome. And also from clusters of such factors. So clusters of these, called piRNA clusters. We know that the noncoding RNA, so the piRNA is bound by PIWI proteins. And in mammals they're known as Mili, Miwi1, and Miwi2, although the other name in mammals is Piwi-like one, or Piwil1, Piwil2, and Piwil4. The important thing is, while there are many PIWI proteins, their roles are non-redundant. In other words, each of the different PIWI proteins has a specific role. So I'll explain the same thing again, but go through with a picture this time. So, we have the transposons, which are shown up here. And the transposons can have unidirectional or bidirectional promoters and they can be clustered or separated out throughout the genome. And they result in, when they're transcribed, they result in piRNA precursors. It's these longer precursors that are then exported from the nucleus. And then undergo primary processing. And primary processing, in this case, just means that they are shortened. They're cut down to slightly shorter lengths, to be that 24 to 35 nucleotides. It's at this point that those those short RNAs are loaded into the PIWI proteins. So this can be the three different types that are found in mammals for example. Mili, MIWI1 and MIWI2. So there's a secondary processing step in addition to that primary processing which is shortening, and this secondary processing event is known as ping pong amplification. And I'm not going to go through the details of ping pong amplification. But all you need to remember about this is that it actually amplifies that piRNA signal in the first place and so, it actually produces more RNA's for silencing the transposable elements than we originally produced. So the first option of what can happen now is that these PIWI protein and piRNA's can result in post transcriptional gene silencing just like happened for microRNA. So in this case we get degradation of the transposon RNA. The transposon RNA would normally be encoding genes that are helping the transposon jump around the genome. So it's a very good thing if you can disable that by cutting up this RNA and therefore, disabling the ability of that transposon to jump. However, perhaps the most interesting feature of the PIWI proteins and the piRNAs that bind to them, is that they can be imported back into the nucleus, and direct DNA methylation. This only happens in the case of MIWI2. So MIWI2. This, this one particular PIWI protein that's found in mammalian cells, can bind to the piRNAs, go back into the nucleus and now through sequence specificity of the piRNA that is found there it will go back and bind to a particular transposon and direct DNA methylation there. So it's this transcriptional silencing, rather than post-transcriptional silencing and the DNA methylation that ensues so that RNA directed DNA methylation, which is one of these unique features of piRNAs. And why there's a lot of interest about piRNA at the moment in epigenetics. So if we compare piRNAs and micro RNAs, we know that micro RNAs require Dicer, this enzyme Dicer, whereas piRNAs do not. We know that micro RNAs only act in post-transcriptional gene silencing. But importantly, piRNAs act in both post-transcriptional gene silencing in a similar way to microRNAs. But also they act in this, they can alter epigenetic state. So they can go back and permanently silence the transposon from which they came through DNA methylation. piRNA's are also important in development and disease. As I mentioned earlier, they're important in the germ line. And also in stem cells. But in addition, and we can see the role in the germ line by knocking out of these various PIWI proteins in mice. So, knock out mice that are deleted for either Mili1 or Mili2 are sterile. And that's because they have impaired spermatogenesis. Oogenesis, so the production of eggs is actually fine, but the production of sperm is compromised. And this is associated with a failure to silence these, silence these repetitive elements. You'll remember I may have mentioned right back when we were talking about DNA methylation intracisternal A particles or IAPs. It's one class of repeats, a common repeat that's found in the mammalian genome. There are also LINE1s and many others. And inappropriate activation of those repeats is associated with infertility in these mice. What's important is that if you find people that have mutations in these PIWI proteins, they are also infertile. And, so clearly this is a broader mechanism. Furthermore, if we look in cancer cells, we can also now find that the PIWI proteins are abhorrently expressed in cancer cells. And so this suggests that they're having a role, not just in the germ line, but also in other contexts. And it's possible that it's the role for PIWI proteins in cancer has some relationship to their role in stem cells So it's something that's going to be, we will be investigating it, or is currently being investigated at the moment. So I've mentioned that these piRNA's are really critical for silencing the transposable elements, the repetitive elements, but do they have a role of silencing other areas in the genome, the non-repetitive regions? What we know is that when you look in a particular type of spermatocyte, you can find the piRNAs are targeting not just the repetitive elements, but also non-repetitive DNA. We can particularly look at one gene, and it's called Rasgrf1. So Rasgrf1 is quite a special gene because it's subject to genomic imprinting. What this means is that it's only expressed from one allele. So, all of your genes you can, you inherit one allele from your mom and one allele from your dad. And normally, both alleles will be expressed. You would use both of them but, in some particular, particularly special cases you only express the copy you inherit from just from your mom or just from your dad. In the case of Rasgrf1 it's paternally imprinted. That means it's silenced on the paternal allele and it's active only from the maternal allele. So you only use the copy that you inherited from your mom. And so this very special class is called genomic imprinting. This class of genes that undergo this monoallelic expression based on the parent of origin and we'll cover this in later lectures on imprinting. But here what's relevant is that this is not a repetitive element. It's not strictly a repetitive element, it's a single copy gene and we know that piRNAs are involved in epigenetic silencing at the single copy gene. And so, while piRNAs are still only very recently being studied, it would appear that they're going to be, that there's still much to be understood and that they may be acting at many more places than just the transposable elements in the genome.
