Mammalian Piwi proteins are important for male germline development particularly spermatogenesis [35
]. In Drosophila
, mutation of Piwi results in sterility and loss of germline cells in males and females [38
]. In vertebrates and invertebrates, expression of Piwi-family proteins is restricted to the germline unlike the ubiquitous expression patterns observed for Argonaute. Until recently the RNAi function of Piwi proteins and the population of small RNAs that they bind to has remained obscure despite the implication that Piwi proteins are functionally similar to Argonautes.
Following immunoprecipitation of Piwi proteins from mammalian testes an abundant population of Piwi-interacting RNAs (piRNAs) were discovered [26
]. Testes-specific mammalian piRNAs are distinctively longer than previously characterized Argonaute associated si- and miRNAs (26−31 nt vs. 21−24 nt). A unique subset of piRNAs in mice distribute between two Piwi proteins, MIWI [26
] and MILI [27
]. MIWI bound piRNAs are slightly longer (29−31 nt) than the MILI bound piRNAs (26−28 nt) but the biological significance of this difference remains unknown. RIWI, the rat homolog of MIWI also binds to 29−30 nt piRNAs in the testes [28
]. Rat and mouse piRNA sequences are extremely diverse and map to their respective genomes in discrete genomic loci. Many piRNAs within a given loci are identified only once and have an extreme strand bias matching either the sense or antisense strand in a non-overlapping manner. In several instances adjacent piRNA loci abruptly switch to the opposing strand [26
]. Sequenced piRNAs have a strong bias for a 5’ uridine similar to repeat-associated siRNAs (rasiRNAs) and miRNAs [39
], although the significance of this bias is unknown.
the previously identified rasiRNAs [39
] have been shown to bind the Piwi family members Piwi and Aubergine (Aub) [40
] and thus represent a subset of Drosophila
piRNAs. Piwi-associated rasiRNAs (now referred to as piRNAs) are longer (24−29 nt) than Argonaute bound siRNAs/miRNAs and are derived predominately from repetitive genomic loci like transposons or Satellite repeats [39
]. The majority of piRNAs matching transposons, was shown to be antisense to the active transposon element [41
]. In flies, loss of Dicer in the miRNA pathway and siRNA pathway has no effect on piRNA levels and/or transposon repression suggesting that Piwi associated small RNAs in Drosophila
have an alternative biogenesis pathway [41
]. The mystery of piRNA biogenesis is becoming clearer with recent sequence data reported for piRNAs interacting with all three Piwi family proteins (Piwi, Aubergine, Ago3) in Drosophila
]. Sequences of piRNAs bound to Piwi and Aubergine are predominately antisense to regions of repeats and transposons in the fly genome similar to previous observations [34
]. Analogous to mammalian piRNAs the Piwi/Aubergine piRNAs also have a strong preference for a 5’ uridine. The surprise came when piRNAs bound to the third Drosophila
Piwi protein (Ago3) were examined. Ago3 piRNAs strongly map to the sense strand orientation and do not exhibit a 5’ uridine bias. Intriguingly the Ago3 associated 5’ end of the sense piRNAs frequently share 10 base pair complementarity with the 5’ end of Aubergine and to a lesser extent Piwi bound antisense piRNAs. Consequently the Ago3 bound piRNAs complementary relationship ensures a strong bias for an adenine at position 10 within the majority of Ago3 piRNA sequences. This observation led Brennecke et al.
] and Gunawardane et al.
] to propose a Slicer mediated mechanism for piRNA biogenesis (). The proposed mechanism requires a pool of primary antisense piRNAs derived potentially from piRNA clusters by an unknown initiation step. Primary antisense piRNAs are loaded into Piwi or Aubergine, which guide targeting of active transposon transcripts. Slicer mediated cleavage between nucleotides 10 and 11 of a target RNA is proposed to yield the 5’ end of sense piRNAs. An unknown endonuclease must then process the 3’ end for loading into Ago3. A sense piRNA/Ago3 complex is poised for targeting complementary piRNA cluster transcripts, where Slicer cleavage by Ago3 builds the 5’ end of antisense piRNA substrate for Piwi/Aubergine complexes. Again 3’ end processing by an endonuclease must assist Ago3, leading to completion of the catalytic cycle. Piwi, Aubergine, and Ago3 each have in vitro
Slicer activity [43
] and are thus capable of catalyzing the reactions in the proposed model. It remains unclear how primary Piwi/Aubergine antisense piRNAs are produced to initiate the amplification loop. Brennecke et al.
suggest that processing of a single stranded piRNA cluster transcript is likely, although maternal supply of piRNA complexes could also serve to initiate the cycle [42
Figure 3 Drosophila piRNA Biogenesis Mechanism. Antisense Piwi-interacting RNAs (piRNAs) in flies associate with Piwi and Aubergine (Aub) proteins. A 10 bp complementary relationship exists for antisense Piwi/Aub piRNAs with sense oriented Ago3 piRNAs, therefore (more ...)
The 3’ processing activity required for the piRNA ping-pong model was recently attributed to two putative nucleases encoded by the zucchini
) and squash
) genes in flies [44
]. Defects in piRNA biogenesis and the upregulation of transposons are evident in zuc
mutant flies. Additionally Zucchini and Squash proteins colocalize and interact directly with Aubergine [44
]. These results strongly implicate these two putative nucleases as the 3’ processing activity in piRNA biogenesis. The current piRNA biogenesis model is based primarily on piRNA sequence data. Further biochemical support for the 5’ and 3’ processing activities responsible for piRNA biogenesis is essential.
Investigation of the relationship of piRNAs bound to the three mouse Piwi proteins (MIWI, MILI, MIWI2) should reveal insight into mammalian piRNA biogenesis. It should be noted that piRNAs are depleted in MIWI [30
] and MILI [45
] mutant mice providing further support for the direct involvement of Piwi proteins in mammalian piRNA biogenesis. As in Drosophila
, evidence for a mammalian amplification loop for 5’ end generation of piRNAs is evident for MILI bound piRNAs [45
] where a similar complimentary relationship between sense and antisense piRNAs are observed.
Transposon control in the germline by Piwi/piRNA complexes may be an evolutionarily conserved function. Characterization of MIWI2 indicates a role in transposon control in the mouse germline. Miwi2
deficient mice have increased transposon activity correlated with decreased DNA methylation [46
mutants also lose DNA methylation in transposons resulting in increased transposon activation [45
In zebrafish, transposon derived piRNAs associate with the zebrafish Piwi protein Ziwi [47
]. As in flies [41
], zebrafish piRNAs are not depleted in Dicer deficient germ cells providing evidence that vertebrate piRNAs are also made by a distinct pathway from siRNA biogenesis. A strong 5’ uridine bias for antisense piRNAs is consistent with the sequenced piRNAs from mammals and flies. For sense oriented piRNAs derived from retroelements there is a tendency for loss of the 5’ uridine bias; however an increased incidence of adenine at position 10 mirrors the trend observed in Drosophila
piRNAs, signifying a conserved biogenesis mechanism may exist for piRNA production in vertebrates. In contrast to genomic piRNA clusters in mammals, zebrafish piRNAs switch strand bias multiple times within a given cluster. This suggests that while a Piwi dependent mechanism of piRNA biogenesis may exist there are key differences between fish and mammals.
In addition to their longer length and unique biogenesis compared to siRNAs, piRNAs carry a 3’ end chemical modification, a feature initially observed in Drosophila
]. Upon examination of the chemical nature of zebrafish piRNAs a similar modification was suggested. Using HPLC and mass spectrometry Houwing et al.
determined that the more abundant rat piRNAs have a 2’-O-methyl modification on the 3’ end [47
] (). Two additional studies on mouse piRNAs using alternative methods report an identical modification [48
]. This modification is also seen in plant miRNAs [50
]. It is mediated by Hen1 methyltransferase and lends stability to the small RNAs. Kirino and Mourelatos more recently describe data showing the mouse homolog of Hen1 contains piRNA methyltransferase activity [51
]. Data from Saito et al. and Horwich et al. now show that the Drosophila
homolog of Hen1 is the piRNA methyl transferase (Pimet) in flies [52
]. It remains to be determined at which point in piRNA biogenesis methylation occurs. It is also not clear what role methylation has on piRNA function. One possibility is that methylation protects piRNAs from 3’−5’ exonucleases in the germline. Alternatively, methylated piRNAs carry a chemical signature that may be specifically recognized by Piwi proteins or other factors.