Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes are mostly involved in the epigenetic and post-transcriptional silencing of transposons, but can also be involved in the regulation of other genetic elements in germ line cells. piRNAs are mostly created from loci that function as transposon traps and provide an RNA-mediated adaptive immunity against transposon expansions and invasions. They are distinct from microRNA (miRNA) in size (26–31 nt rather than 21–24 nt), lack of sequence conservation, and increased complexity. Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes are mostly involved in the epigenetic and post-transcriptional silencing of transposons, but can also be involved in the regulation of other genetic elements in germ line cells. piRNAs are mostly created from loci that function as transposon traps and provide an RNA-mediated adaptive immunity against transposon expansions and invasions. They are distinct from microRNA (miRNA) in size (26–31 nt rather than 21–24 nt), lack of sequence conservation, and increased complexity. In 2008, it was still unclear how piRNAs are generated, but potential methods had been suggested, and it was certain their biogenesis pathway is distinct from miRNA and siRNA, while rasiRNAs are a piRNA subspecies. piRNAs have been identified in both vertebrates and invertebrates, and although biogenesis and modes of action do vary somewhat between species, a number of features are conserved. piRNAs have no clear secondary structure motifs, the length of a piRNA varies between species, from 21 to 31 nucleotides, and the bias for a 5’ uridine is common to piRNAs in both vertebrates and invertebrates. piRNAs in Caenorhabditis elegans have a 5’ monophosphate and a 3’ modification that acts to block either the 2’ or 3’ oxygen, and this has also been confirmed to exist in Drosophila melanogaster, zebrafish, mice and rats. This 3’ modification is a 2’-O-methylation; the reason for this modification is not clear, but it has been suggested to increase piRNA stability.It is thought that there are many hundreds of thousands of different piRNA species found in mammals. Thus far, over 50,000 unique piRNA sequences have been discovered in mice and more than 13,000 in D. melanogaster. In the early 1980s, it was discovered that in the fruit fly genome a single mutation could specifically activate all copies of a retrovirus-like element called Gypsy in the female germline. The site of these mutations that made these Gypsies 'dance' was thus called the flamenco locus. In 2001, Aravin et al. proposed that double-stranded (ds) RNA-mediated silencing is implicated in the control of retrotransposons in the germline and by 2003 the idea originated that vestiges of transposons might produce dsRNAs required for the silencing of 'live' transposons. Sequencing of the 200,000 bp flamenco locus was hard, as it turned out to be packed with transposable element fragments (104 insertions of 42 different transposons, including multiple Gypsies), all facing the same direction. Indeed, piRNAs are all found in clusters throughout animal genomes; these clusters may contain as few as ten or up to thousands of piRNAs matching different, phased transposon fragments. This led to the idea in 2007 that in germlines a pool of primary piRNAs is processed from long single-stranded transcripts encoded by piRNA clusters in the opposite orientation of the transposons, so that the piRNAs can anneal to and target the transposon encoded transcripts, thereby triggering their degradation. Any transposon landing in the correct orientation in such a cluster will make the individual more or less immune to that transposon, and such an advantageous mutation will spread quickly through the population. The original mutations in the flamenco locus inhibited the transcription of the master transcript, thereby deactivating this defense system. A historical example of invasion and Piwi response is known: the P-element transposon invaded a fruit fly genome in the mid-20th century, and, through interbreeding, within decades all wild fruit flies worldwide (but not the isolated lab strains) contained the element. Repression of further P-element acitivy, spreading near-simultaneously, appears to have been by the Piwi-interacting RNA pathway. piRNA clusters in genomes can now readily be detected via bioinformatics methods. While D. melanogaster and vertebrate piRNAs have been located in areas lacking any protein coding genes, piRNAs in C. elegans have been identified amidst protein coding genes. In mammals, piRNAs are found both in testes and ovaries, although they only seem to be required in males. In invertebrates, piRNAs have been detected in both the male and female germlines. At the cellular level, piRNAs have been found within both nuclei and cytoplasm, suggesting that piRNA pathways may function in both of these areas and, therefore, may have multiple effects. The biogenesis of piRNAs is not yet fully understood, although possible mechanisms have been proposed. piRNAs show a significant strand bias, that is, they are derived from one strand of DNA only, and this may indicate that they are the product of long single stranded precursor molecules. A primary processing pathway is suggested to be the only pathway used to produce pachytene piRNAs; in this mechanism, piRNA precursors are transcribed resulting in piRNAs with a tendency to target 5’ uridines. Also proposed is a ‘Ping Pong’ mechanism wherein primary piRNAs recognise their complementary targets and cause the recruitment of piwi proteins. This results in the cleavage of the transcript at a point ten nucleotides from the 5’ end of the primary piRNA, producing the secondary piRNA. These secondary piRNAs are targeted toward sequences that possess an adenine at the tenth position. Since the piRNA involved in the ping pong cycle directs its attacks on transposon transcripts, the ping pong cycle acts only at the level of transcription. One or both of these mechanisms may be acting in different species; C. elegans, for instance, does have piRNAs, but does not appear to use the ping pong mechanism at all. A significant number of piRNAs identified in zebrafish and D. melanogaster contain adenine at their tenth position, and this has been interpreted as possible evidence of a conserved biosynthetic mechanism across species. Ping-pong signatures have been identified in very primitive animals such as sponges and cnidarians, pointing to the existence of the ping-pong cycle already in the early branches of metazoans.