Mighty Piwis Defend the Germline against Genome Intruders
2007; Cell Press; Volume: 129; Issue: 1 Linguagem: Inglês
10.1016/j.cell.2007.03.028
ISSN1097-4172
AutoresKathryn A. O’Donnell, Jef D. Boeke,
Tópico(s)Animal Genetics and Reproduction
ResumoPiwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Mobile elements can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Rampant mobility of these elements would endanger both the host and, thereby, the element. Thus a strong selective pressure exists to limit their transposition. Mobile elements are classified into two categories based on the mechanism of their transposition. DNA transposons, such as Drosophila P elements, generally utilize a cut-and-paste mechanism in which the transposon is excised from a donor site and inserted into a new genomic location. Retrotransposons and endogenous retroviruses such as gypsy elements represent a distinct class of mobile genetic sequences that insert into new genomic locations by reverse transcription of an RNA intermediate. Expansion of these selfish elements can occur when novel transposition events are transmitted to subsequent generations after germline hopping; indeed metazoan transposons often show germline-restricted expression. Therefore, it seems likely that metazoan genomes have evolved mechanisms to regulate germline mobilization of transposable elements. DNA methylation is one important mechanism involved in the silencing of transposons in plant, mammalian, and fungal germlines (Yoder et al., 1997Yoder J.A. Walsh C.P. Bestor T.H. Cytosine methylation and the ecology of intragenomic parasites.Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1450) Google Scholar, Martienssen and Colot, 2001Martienssen R.A. Colot V. DNA methylation and epigenetic inheritance in plants and filamentous fungi.Science. 2001; 293: 1070-1074Crossref PubMed Scopus (383) Google Scholar, Selker, 2004Selker E.U. Genome defense and DNA methylation in Neurospora.Cold Spring Harb. Symp. Quant. Biol. 2004; 69: 119-124Crossref PubMed Scopus (22) Google Scholar). Additionally, APOBECs (a class of RNA/DNA-editing enzymes) have been found to be potent genome defense proteins against retroelements (Takaori-Kondo, 2006Takaori-Kondo A. APOBEC family proteins: novel antiviral innate immunity.Int. J. Hematol. 2006; 83: 213-216Crossref PubMed Scopus (20) Google Scholar). RNAi is widely believed to control retrotransposition (Robert et al., 2004Robert V.J. Vastenhouw N.L. Plasterk R.H. RNA interference, transposon silencing, and cosuppression in the Caenorhabditis elegans germ line: similarities and differences.Cold Spring Harb. Symp. Quant. Biol. 2004; 69: 397-402Crossref PubMed Scopus (19) Google Scholar); however, this system has a surprisingly modest effect on silencing mammalian retrotransposons in somatic cells (Yang and Kazazian, 2006Yang N. Kazazian Jr., H.H. L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells.Nat. Struct. Mol. Biol. 2006; 13: 763-771Crossref PubMed Scopus (297) Google Scholar). With the recent characterization of the molecular function of Piwi (P element-induced wimpy testes) proteins, a novel form of control for mobile elements has emerged involving small RNAs in germ cells. The founding member of this class of proteins, Piwi, was first identified 10 years ago in a genetic screen for mutants that affect asymmetric division of stem cells in the Drosophila germline (Lin and Spradling, 1997Lin H. Spradling A.C. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.Development. 1997; 124: 2463-2476Crossref PubMed Google Scholar). Early studies demonstrated that Drosophila Piwi is essential for spermatogenesis and is a key regulator of female germline stem cells (Cox et al., 2000Cox D.N. Chao A. Lin H. piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells.Development. 2000; 127: 503-514PubMed Google Scholar). It was also appreciated that Piwi proteins are an ancient subset of the larger Argonaute protein family (Carmell et al., 2002Carmell M.A. Xuan Z. Zhang M.Q. Hannon G.J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis.Genes Dev. 2002; 16: 2733-2742Crossref PubMed Scopus (668) Google Scholar, Cerutti and Casas-Mollano, 2006Cerutti H. Casas-Mollano J.A. On the origin and functions of RNA-mediated silencing: from protists to man.Curr. Genet. 2006; 50: 81-99Crossref PubMed Scopus (359) Google Scholar), other members of which associate with short-interfering (si)RNAs and micro (mi)RNAs. These small RNAs serve as guides that lead to degradation and/or reduced translation of target mRNAs. Membership in the Argonaute family suggested that Piwi proteins and their associated RNAs might also mediate RNA silencing. The recent identification and characterization of the small Piwi-interacting RNAs (dubbed piRNAs) has indicated that Piwi proteins mediate RNA-mediated silencing of mobile elements, thereby defending the germline genome. The murine Piwi orthologs Miwi and Mili are essential for mammalian spermatogenesis (Deng and Lin, 2002Deng W. Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis.Dev. Cell. 2002; 2: 819-830Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, Kuramochi-Miyagawa et al., 2004Kuramochi-Miyagawa S. Kimura T. Ijiri T.W. Isobe T. Asada N. Fujita Y. Ikawa M. Iwai N. Okabe M. Deng W. et al.Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.Development. 2004; 131: 839-849Crossref PubMed Scopus (572) Google Scholar). Mice with targeted mutations in either gene are sterile and have distinct defects in gametogenesis, but unlike the Drosophila piwi mutant, neither loses germline stem cells. To investigate the role of the third mouse Piwi family member in gametogenesis, the gene encoding Miwi2 has now been disrupted. In a report described in Developmental Cell, Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar demonstrate that Miwi2 mutants are unique in their loss of germline stem cells. These observations suggest that the stem cell maintenance functions exhibited by Drosophila Piwi are conserved in mice through the function of Miwi2. After initial characterization of the MILI/MIWI proteins in mice, the next challenge was to identify their small RNA-binding partners. Last year, five independent laboratories reported the identification of mammalian piRNAs from mouse and rat testes (Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. et al.A novel class of small RNAs bind to MILI protein in mouse testes.Nature. 2006; 442: 203-207Crossref PubMed Scopus (1010) Google Scholar, Girard et al., 2006Girard A. Sachidanandam R. Hannon G.J. Carmell M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature. 2006; 442: 199-202Crossref PubMed Scopus (1112) Google Scholar, Grivna et al., 2006Grivna S.T. Beyret E. Wang Z. Lin H. A novel class of small RNAs in mouse spermatogenic cells.Genes Dev. 2006; 20: 1709-1714Crossref PubMed Scopus (602) Google Scholar, Lau et al., 2006Lau N.C. Seto A.G. Kim J. Kuramochi-Miyagawa S. Nakano T. Bartel D.P. Kingston R.E. Characterization of the piRNA complex from rat testes.Science. 2006; 313: 363-367Crossref PubMed Scopus (690) Google Scholar, Watanabe et al., 2006Watanabe T. Takeda A. Tsukiyama T. Mise K. Okuno T. Sasaki H. Minami N. Imai H. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes.Genes Dev. 2006; 20: 1732-1743Crossref PubMed Scopus (445) Google Scholar). Two of these groups purified ribonucleoprotein complexes with a MILI- or MIWI-specific antibody from adult mouse testes and then cloned and sequenced the associated small RNAs. These MILI- and MIWI-interacting RNAs were termed piRNAs based on their interaction with the mouse Piwi proteins. piRNAs have several interesting characteristics. First, these small RNAs were longer than miRNAs and siRNAs and similar in size to a previously described class of Drosophila RNAs corresponding to repeat sequences, “rasiRNAs” (repeat-associated siRNAs; Aravin et al., 2003Aravin A.A. Lagos-Quintana M. Yalcin A. Zavolan M. Marks D. Snyder B. Gaasterland T. Meyer J. Tuschl T. The small RNA profile during Drosophila melanogaster development.Dev. Cell. 2003; 5: 337-350Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Second, the majority of piRNAs mapped to a small number of genomic loci. Individual clusters range between 1 and 100 kb in size and contain between 10 and 4500 piRNAs, demonstrating that thousands of piRNAs may be generated from one particular locus. Third, many of these clusters exhibit remarkable asymmetry, meaning that within a given cluster all piRNAs are derived from the same strand. This asymmetric orientation suggests that piRNAs might be processed from long primary transcripts. When two adjacent clusters were located in close proximity to each other, strand switching was also commonly observed. Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. et al.A novel class of small RNAs bind to MILI protein in mouse testes.Nature. 2006; 442: 203-207Crossref PubMed Scopus (1010) Google Scholar postulated that these neighboring clusters with opposite strand polarity might be transcribed divergently from one bidirectional promoter. Sequence analysis of the MILI- and MIWI-associated piRNAs revealed a strong bias for uridine residues at their 5′ termini. This 5′ uridine bias is characteristic of siRNAs and miRNAs processed from double-stranded precursors by RNase III enzymes. However, a computational search for stem loops similar to pre-miRNAs failed to identify any secondary structures in regions flanking piRNAs, suggesting that piRNA processing is distinct from miRNA biogenesis. Finally, ∼17% of mammalian piRNAs mapped to repeat sequences, including LINEs, SINEs, and several classes of DNA transposons. Although this is consistent with a possible role in mobile element defense, considering that ∼40% of the mouse genome is composed of repetitive elements, this is actually less than expected by chance. However, a conserved role for Miwi2 in mobile element control is suggested by the observation of increased L1 retrotransposon expression in the Miwi2 mutant testes (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Interestingly, this increase in L1 transcription was accompanied by decreased L1 DNA methylation, suggesting a possible interplay between Piwi (and perhaps piRNAs) and methylation machinery, reminiscent of the interaction between the siRNA posttranscriptional silencing machinery and chromatin level transcriptional regulation in Schizosaccharomyces pombe (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (911) Google Scholar). However, this analogy notwithstanding, it is important to note that no Miwi2-specific piRNAs have yet been described, so it is formally possible that this pathway is piRNA independent. This raises the question, how pervasive is the Piwi-piRNA-genome defense association? In several of the earlier piRNA sequence studies, the majority of piRNAs were identified only once, suggesting a high degree of complexity in piRNA populations. Comparative genomics further revealed that the piRNA loci, but not their sequences, are conserved throughout evolution. As Girard et al., 2006Girard A. Sachidanandam R. Hannon G.J. Carmell M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature. 2006; 442: 199-202Crossref PubMed Scopus (1112) Google Scholar point out, this may indicate that the sequence of a piRNA does not necessarily specify its function. Rather, its true function may be determined by the abundance of piRNAs produced from any individual locus. Despite these interesting and confounding discoveries, several important questions remained. Do piRNAs exist in invertebrates and other vertebrate species? What are their mRNA targets? Are piRNAs similar to Drosophila rasiRNAs? Is there more compelling evidence that piRNAs provide defense against genome intruders like mobile elements? Two new papers, one in this issue of Cell (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar), shed light on some of these questions and provide us with more food for thought. Although piRNAs were first identified in mammals, analogous studies in flies revealed that this class of small RNAs also exists in invertebrates. Recently, two Piwi family members in Drosophila, Aubergine and Piwi, were found to bind small RNAs (Saito et al., 2006Saito K. Nishida K.M. Mori T. Kawamura Y. Miyoshi K. Nagami T. Siomi H. Siomi M.C. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome.Genes Dev. 2006; 20: 2214-2222Crossref PubMed Scopus (459) Google Scholar, Vagin et al., 2006Vagin V.V. Sigova A. Li C. Seitz H. Gvozdev V. Zamore P.D. A distinct small RNA pathway silences selfish genetic elements in the germline.Science. 2006; 313: 320-324Crossref PubMed Scopus (900) Google Scholar). In a study reporting a few hundred piRNA sequences, Saito et al. demonstrated that Piwi complex immunopurified from Drosophila ovaries contained a class of small RNAs distinct in size from siRNAs and miRNAs. Sequencing revealed that most of these piRNAs corresponded to repetitive elements and heterochromatic genome regions. Tuschl and colleagues had previously identified about 4000 Drosophila germline rasiRNAs (Aravin et al., 2003Aravin A.A. Lagos-Quintana M. Yalcin A. Zavolan M. Marks D. Snyder B. Gaasterland T. Meyer J. Tuschl T. The small RNA profile during Drosophila melanogaster development.Dev. Cell. 2003; 5: 337-350Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), which also corresponded to repetitive elements, suggesting that they might regulate chromatin structure and transposon activity. Based on current evidence, it appears that most rasiRNAs in flies are simply a (very important) subclass of piRNAs. In recent years, Piwi proteins were recognized as having potential anti-mobile element activity. Transposition of telomeric retroelements and P elements is enhanced in aubergine mutants whereas piwi mutants mobilized the endogenous retrovirus gypsy (Sarot et al., 2004Sarot E. Payen-Groschene G. Bucheton A. Pelisson A. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.Genetics. 2004; 166: 1313-1321Crossref PubMed Scopus (187) Google Scholar) and showed increased expression of copia and mdg1 elements (Kalmykova et al., 2005Kalmykova A.I. Klenov M.S. Gvozdev V.A. Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline.Nucleic Acids Res. 2005; 33: 2052-2059Crossref PubMed Scopus (168) Google Scholar). Vagin et al., 2006Vagin V.V. Sigova A. Li C. Seitz H. Gvozdev V. Zamore P.D. A distinct small RNA pathway silences selfish genetic elements in the germline.Science. 2006; 313: 320-324Crossref PubMed Scopus (900) Google Scholar also demonstrated that expression of retrotransposons was derepressed in the germline of piwi and aubergine mutants. Importantly, silencing of these retroelements did not require RNAi or miRNA proteins. These findings suggested that Piwi proteins and their associated small RNAs might silence mobile elements in the germline. In a recent issue of Cell, Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar investigate the small RNA-binding partners of Piwi, Aubergine, and Ago3 in the Drosophila female germline at high resolution. After purifying RNP complexes using antibodies specific to each of the three proteins, cDNA libraries were prepared from each of the piRNA populations. 454 sequencing yielded more than 60,000 piRNA reads, providing a much larger sequence population to analyze than in the earlier fly studies. Similar to mammalian piRNAs, Drosophila piRNAs are longer than miRNAs and siRNAs and map to discrete genomic clusters. For example, the largest 15 clusters account for 70% of all piRNAs, suggesting that a limited number of master piRNA loci might control germline mobile element activity. Unlike their mammalian counterparts, most piRNAs in flies (∼80%) are present in pericentromeric and telomeric heterochromatin and correspond to truncated or defective repeat elements. How do these findings square with earlier studies of transposon control mechanisms? One model of transposon control proposes that transposon resistance is due to discrete genomic loci and is supported by studies of the gypsy element, the first endogenous retrovirus discovered in invertebrates. The mobility of gypsy and two other retroelements, Idefix and ZAM, is controlled by flamenco, a specific heterochromatic locus in the X chromosome (Bucheton, 1995Bucheton A. The relationship between the flamenco gene and gypsy in Drosophila: how to tame a retrovirus.Trends Genet. 1995; 11: 349-353Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Despite intensive study of flamenco, no “transposon repressor locus” could be identified in the sequence. Rather, it contained a jumble of different types of transposable elements, but exactly how these elements might be involved in a transposon defense system remained unclear. Sarot et al., 2004Sarot E. Payen-Groschene G. Bucheton A. Pelisson A. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.Genetics. 2004; 166: 1313-1321Crossref PubMed Scopus (187) Google Scholar provided one connection by showing that flamenco-mediated silencing of gypsy depends on Piwi. Now, Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar provide direct sequence evidence that a large piRNA locus spanning more than 150 kb corresponds to the flamenco locus. The depth of the sequencing allowed them to find many instances of mobile element-derived piRNAs mapping uniquely to flamenco. Further supporting the notion that piRNA clusters are control loci that regulate transposon activity through the Piwi pathway, Brennecke et al. performed several functional tests using flamenco mutants. In agreement with their hypothesis, mature piRNA expression levels decreased in flamenco mutants whereas gypsy mRNA expression increased. Brennecke et al. also demonstrate that the subtelomeric TAS repeat on the X chromosome (X-TAS) corresponds to yet another piRNA cluster. Previous studies have linked specific alleles of this locus, here designated X-TASP, to the global control of P elements (see references in Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar). Those alleles are distinguished by containing P element insertions in X-TAS. The sites from which piRNAs (not complementary to P elements) emanate in the Oregon R fly strain analyzed by Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar correspond to the insertion positions of three P elements found in a series of X-TASP strains. The Oregon R fly strain does not contain P sequences at X-TAS. Thus it seems likely that the X-TASP loci will produce P element-derived piRNAs. This is truly remarkable because P elements invaded the D. melanogaster genome only within the last 50 years, presumably sweeping in through contact with a sibling species (Kidwell, 1983Kidwell M.G. Evolution of hybrid dysgenesis determinants in Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 1983; 80: 1655-1659Crossref PubMed Scopus (212) Google Scholar). The implication is that the resistance locus was born when P elements inserted into X-TAS, within very recent history, showing how dynamic the interplay between host and genome parasite can be even on a short time scale. A model for piRNA-mediated suppression of transposons is shown in Figure 1. Using flamenco and X-TAS as examples, these heterochromatic loci generate hundreds of distinct piRNAs that correspond to transposon repeats dispersed throughout the Drosophila genome. These piRNAs associate with Piwi proteins and serve as guides that lead to cleavage of expressed transposon targets. By examining the strand bias of piRNAs derived from each of the three Piwi complexes, these authors, as well as Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar who performed a smaller piRNA sequencing study, made several other important observations consistent with a genome defense mechanism. Piwi and Aubergine preferentially bind piRNAs corresponding to the antisense strand of transposons. In contrast, Ago3 complexes are biased for the sense strand of transposons. Perhaps one of the most intriguing findings is the observation of a unique complementary relationship between these sense and antisense piRNAs. Assuming that piRNAs are ∼25 nucleotides long, one would expect corresponding sense and antisense piRNAs to overlap by 23 nucleotides with a 2 nucleotide 3′ overhang at each end if processed in an siRNA- or miRNA-like manner. In fact, this was not observed with complementary piRNAs. Instead, the 5′ ends of complementary piRNAs were separated by 10 nucleotides, with the strongest complementarity observed between Ago3- and Aubergine-associated piRNAs. Yet another surprise was the enrichment of 5′-terminal uridine residues in Piwi- and Aubergine-bound piRNAs, which correspond to the antisense strand of transposons. As might be expected for sense strand piRNAs bound to Ago3, these show a dramatic enrichment for adenine at position 10, and they complement the 5′-terminal uridine of an antisense piRNA bound to Piwi or Aubergine. Notably, the same strand bias was observed for piRNAs bound to Drosophila Piwi proteins (Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar). These findings suggest that Piwi-mediated cleavage events generate new piRNAs. In light of these findings, both groups propose a self-reinforcing amplification cycle for piRNA generation that may be analogous to secondary siRNA generation by RNA-dependent RNA polymerases (Figure 2). According to this model, initiation of the cycle begins with processing of primary piRNAs, which are derived from defective transposon copies in regions of heterochromatin. These piRNAs are antisense to expressed transposons and bind either Piwi or Aubergine. Together, the Piwi/Aubergine-piRNA complexes identify and cleave their active transposon targets, generating new sense piRNAs that bind Ago3. Next, a sense piRNA-Ago3 complex directs another cleavage event of a piRNA cluster transcript, creating a new antisense piRNA capable of binding to Piwi or Aubergine. Amplification of the response is dependent on the interaction between piRNA sequences in different clusters. As long as secondary antisense piRNA complexes are able to recognize and silence their target transposable elements, the cycle is reinforced through the production of additional sense piRNAs. Although several aspects of the model have yet to be validated, this amplification loop has important implications for mobile element control in the germline. The proposed model raises several important questions. How is the amplification cycle initiated with primary antisense piRNAs loaded into Piwi or Aubergine? Although it is logical that Ago3-bound piRNAs would be in the sense orientation if they were generated solely by piRNA-mediated cleavage of transposon sequences, the origin of the strict antisense strand bias of Piwi- and Aubergine-bound piRNAs is not intuitive. Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar demonstrate that there are special loci such as flamenco from which piRNAs are generated from only one strand and specificall
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