Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. The role of aubergine in oskar translation has been examined. aubergine has been implicated in dorsoventral patterning, since eggs from aubergine mutant mothers are ventralized and seldom fertilized (Schüpbach, 1991). Two new alleles of aubergine have been isolated in a novel genetic screen. aubergine has been shown to also be required for posterior body patterning, since the small fraction of eggs from aubergine minus mothers that are fertilized develop into embryos that lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either oskar mRNA or protein, the level of Oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance oskar translation. While aubergine-dependence is conferred upon oskar mRNA by sequences in the oskar 3' UTR, aubergine may influence oskar translation through an interaction with sequences upstream of the oskar 3' UTR (Wilson, 1996).
The characterization of maternal-effect mutants that produce embryos with abdominal defects led to the discovery of the 'posterior group' genes, which act in posterior body patterning. Additional maternally acting genes are likely to participate in this process, but they may not have been identified as posterior group genes because they function at multiple stages of development, with the resulting phenotypes obscuring any posterior patterning defects. An approach to identify such genes was suggested by the finding that the bicaudal phenotype resulting from over- or mis-expression of osk could be partially suppressed if the mothers were also heterozygous for a mutation in one of the posterior group genes, such as vas or tud. Similarly, the BicaudalD phenotype is suppressed in vas minus and tud minus heterozygotes. Apparently, low levels of these proteins are limiting in the formation of bicaudal embryos. A screen for mutations which, when heterozygous, partially suppresses the formation of bicaudal embryos might therefore uncover additional genes involved in posterior body patterning. Such a screen was performed using the OB1 transgene to produce a bicaudal phenotype (Wilson, 1996).
The OB1 transgene consists of a slightly modified osk coding region fused to the bcd 3' UTR. Since the bcd 3' UTR contains the bcd mRNA localization signal, the encoded Osk protein is misexpressed at the anterior pole of the oocyte and embryo. Among the mutants recovered were two new alleles of aubergine. aub has been identified as one of a class of female-sterile mutants in which the eggshell is ventralized to variable extents (Schüpbach, 1991). Eggs laid by these mutants are typically not fertilized and consequently do not secrete cuticles, making it difficult to detect any additional patterning defects. The partial suppression of the bicaudal phenotype in aub minus heterozygotes led to an examination of the eggs laid by aub minus mothers (for simplicity, embryos are referred to by the genotype of their mothers). Approximately 2% of aub mutant eggs are fertilized. All of the resulting embryos lack abdominal segments, the classic posterior group mutant phenotype. (At least half of these cuticles display additional abnormalities, which presumably reflect substantial defects in both dorsoventral and anteroposterior body patterning). Thus aub is required for posterior patterning of the embryo. The fertilized eggs of aub mutant mothers lack pole cells; the same is true for most other posterior group mutants (Wilson, 1996).
Two events in which aub acts in posterior body patterning, and one in which aub acts in dorsoventral body patterning, have been documented. Considered in most detail is the enhancement of osk translation by aub. Curiously, the effect of aub mutations on osk translation does not appear to be related to the partial suppression of the OB1 bicaudal phenotype (Wilson, 1996).
A simple explanation for the action of aub in anteroposterior and dorsoventral patterning is that aub plays a role in a process that these two systems utilize in common. mRNA localization is one such process. Therefore the distributions of several localized mRNAs were compared in wild-type and aub mutant ovaries. The distributions of bcd and grk mRNA are wild-type in aub mutants. The early phases of osk mRNA localization, including concentration in the oocyte of early egg chambers and the initial localization to the posterior pole during stages 7-8, are also not affected by aub mutations. In contrast, aub mutants are defective in maintenance of osk mRNA localization. Normally, osk mRNA remains tightly localized to the posterior cortex of later-staged oocytes and early embryos. In aub mutants, osk mRNA prematurely diffuses from the posterior of oocytes and can only rarely be detected at the posterior of embryos (Wilson, 1996).
Although the results presented above could suggest that aub acts differently in anteroposterior and dorsoventral patterning, it is also possible that the primary role is in a single type of process, and that the requirement for aub in osk mRNA maintenance is indirect. One possibility is that aub mutants are defective in the accumulation of specific proteins. One affected protein would be Osk, since Osk protein acts to maintain osk mRNA localization, and another would be a protein required for dorsoventral patterning. To test this idea, the levels of Osk protein were compared in wild type and aub mutants. Despite normal amounts of osk mRNA, the levels of Osk protein are substantially reduced. At least two protein products of the osk gene are detected on Western blots: an abundant protein of approximately 55,000 Mr and a rarer protein of about 70,000 Mr. The amounts of both Osk proteins are decreased in aub mutants, although the 55,000 Mr protein is affected to a greater degree. Immunohistochemistry was used to monitor Osk protein. Under normal staining conditions, very little Osk protein is detected in aub mutant ovaries, and its appearance is slightly delayed relative to wild-type. By extending the staining reaction some Osk protein was detected in almost all aub mutant ovaries, with the protein appearing dispersed over the posterior third of the oocyte, rather than tightly concentrated at the posterior cortex as in wild-type ovaries (Wilson, 1996).
The level of Grk protein is also greatly reduced in aub mutants, while the level of grk mRNA remains normal. This decrease in the amount of Grk protein may give rise to the dorsoventral patterning defects of aub mutants. Nevertheless, the Grk protein present in aub mutants is apparently sufficient for establishment of correct anteroposterior polarity, since aub mutants do not exhibit general defects in mRNA localization or in migration of the oocyte nucleus to the anterior, two processes which rely on grk. In addition, aub is not generally required for the accumulation of proteins during oogenesis, since aub mutations do not affect the levels of any of several other ovarian proteins tested, including Vas, Exu, Kel, and Sn (Wilson, 1996).
Because osk mRNA levels remain normal in aub mutants, but Osk protein is reduced, aub must influence either the synthesis or stability of Osk protein. Evidence that argues strongly against a role for aub in stabilizing Osk protein comes from experiments employing the OB1 transgene. Osk protein is normally detected at both poles of OB1 transgenic embryos; the anterior protein is from the transgene, while the posterior protein is from the endogenous osk gene. In aub;OB1 embryos, the posterior Osk protein is largely missing, however, the anterior Osk protein encoded by the OB1 mRNA remains present at a high level. A similar effect is found in aub;OB1 ovaries. To rule out the possibility that the slight modification of the Osk protein encoded by the OB1 transgene affects the accumulation of anterior Osk protein in aub mutants, these experiments were repeated using the mob transgene. Like OB1, this transgene misexpresses Osk protein at the anterior pole of oocytes and embryos. However, mob encodes a wild-type Osk protein. The distributions of osk protein in aub;mob ovaries and embryos is indistinguishable from that in aub;OB1 animals (Wilson, 1996).
Because the proteins encoded by osk and mob are identical (the genes differ only in their 3' UTRs), aub is unlikely to affect the stability of Osk protein. Thus, aub appears to enhance translation of osk mRNA (Wilson, 1996).
The differential dependence of osk and OB1 mRNAs on aub for translation also provides some insight into which features confer aub-dependence on the osk mRNA. Because the mRNAs differ only in their 3' UTRs, it can be concluded that the requirement for aub is conferred by the osk mRNA 3' UTR. Further results suggest that aub may interact, directly or indirectly, with sequences outside of the osk 3' UTR to influence translation. While translation of osk mRNA is reduced in aub mutants, translation of the OB1 message is increased. Although only low levels of Osk protein are detected in the oocytes of early-staged, OB1 egg chambers, high levels of Osk protein are seen when these transgenic ovaries are additionally mutant for aub. Furthermore, the few, small patches of anteriorly localized Osk protein that are first detected in OB1 egg chambers in stages 7 and 8 are expanded to outline the anterior margin of aub;OB1 egg chambers. This effect is both substantial and highly reproducible. It is unlikely that the increased translation of OB1 mRNA in aub mutants is mediated through the bcd 3' UTR, which is present in the OB1 message, since the levels of Bcd protein (determined by whole-mount immunostaining and Western analysis) are not affected by aub mutations. Instead, wild-type aub function appears to modestly inhibit the translation of the hybrid message, and this effect must be mediated by sequences in the osk mRNA 5' UTR or coding region (Wilson, 1996).
Bruno protein binds sequences in the osk 3' UTR termed BREs and represses osk translation. One potential role for aub, as a positive regulator of osk translation, could be to override this known form of repression. If so, an osk mRNA not repressed by Bruno would not require aub for the normal high levels of translation. To test this possibility, the P[oskBRE minus] transgene, which differs from wild-type osk only by point mutations in the BREs, was used. The P[oskBRE minus] and control P[osk plus ] transgenes were introduced into aub minus flies. Comparing the levels of Osk protein in aub; P[oskBRE minus ] and aub; P[osk plus] ovaries by Western analysis, no difference was found. Thus the absence of Bruno-mediated repression does not eliminate the requirement for aub in osk translation (Wilson, 1996).
These results clearly implicate aub in translation of osk. However, they do not fully explain the posterior body patterning defects caused by the absence of aub for two reasons. (1) The reduced levels of Osk protein found in aub mutants might not be expected to eliminate abdominal segmentation completely, since Osk protein normally appears to be present in substantial excess. Instead, the dispersal of Osk protein across much of the posterior third of the oocyte in aub mutants might be expected to cause ectopic posterior body patterning. Nevertheless, there is little or no abdominal segmention in embryos from aub minus mothers. (2) While the OB1 bicaudal phenotype is partially suppressed in aub minus heterozygotes, translation of the OB1 transgene does not require aub: anterior Osk protein accumulates in OB1 ovaries (and also in embryos), even when both alleles of aub are mutant (Wilson, 1996).
An explanation for the extreme posterior patterning defects of aub mutants, and the partial suppression of the OB1 bicaudal phenotype in aub minus heterozygotes, is revealed by further analysis of embryos from OB1 or aub;OB1 mothers. In OB1 embryos, anterior Osk protein ectopically activates nos and results in the production of bicaudal embryos. Although anterior Osk protein is also present in aub;OB1 embryos, there is no corresponding anterior recruitment of nos mRNA or accumulation of Nos protein, despite normal levels of nos mRNA (Wilson, 1996).
Not surprisingly, the resulting embryos lack abdominal segmentation. Therefore aub is also required for a second event in posterior body patterning, intermediate between osk mRNA translation and nos mRNA localization. Again, this event might involve translational regulation. Candidate mRNAs for this second regulatory event are encoded by the vas and tud genes, both of which are conventionally portrayed as intermediate between osk and nos in the posterior body patterning hierarchy and are also required for the activity of anteriorly localized Osk protein. By Western analysis, however, both proteins remain at wild-type levels in aub mutant ovaries. Consequently, the second requirement for aub in posterior body patterning remains incompletely defined (Wilson, 1996).
Vasa (Vas), a key protein in establishing the specialized translational activity of the Drosophila pole plasm, accumulates at the posterior pole of the developing oocyte. Mutation in gustavus (gus), a gene that encodes a protein that interacts with Vas, blocks posterior localization of Vas, as does deletion of a segment of Vas containing the GUS binding site. Like Vas, Gus is present in cytoplasmic ribonucleoprotein particles. Heterozygotes for gus or a deletion including gus produce embryos with fewer pole cells and posterior patterning defects. Therefore, Gus is essential for the posterior localization of Vas. However, gus is not required for the posterior localization of oskar (osk). The effects of the gusZ409 mutation on pole plasm assembly can mostly be explained through the failure of Vas to be deployed to the posterior pole plasm in this mutant. An exception, however, is the striking reduction of Osk level at the posterior of stage 10 gusZ409 oocytes, since vas alleles have little if any effect on Osk accumulation at this developmental stage. This observation can be explained if gus also affects the spatial distribution of Aub, a translational activator of osk that is a component of polar granules and probably of nuage particles. Posterior accumulation of Aub is vas dependent and therefore is presumably dependent on gus, but Aub-mediated translational activation of osk does not require it to be posteriorly deployed. However, if Aub is a component of nuage particles like Vas, which is suggested by the observation that vas mutations largely abolish perinuclear accumulation of Aub-containing particles in nurse cells, then the gusZ409 mutation could delay or block the movement of Aub through the ring canals and into the oocyte cytoplasm and render it less able to activate translation of osk, thus explaining the observation of decreased Osk at the posterior (Styhler, 2002).
Identification of Aub as a polar granule component suggests that it, like other such components, may be required for pole cell formation. Although aub mutants lack pole cells, this could be attributed to their deficiency in synthesis of Osk protein, a prerequisite for pole cell formation. Therefore, an osk-bcd transgene was used to bypass the requirement for aub in osk mRNA translation and to assess the role of aub in pole cell formation. In this transgene, the osk 3'UTR is replaced with the bicoid (bcd) localization signal, and the encoded mRNA is localized to the anterior of the oocyte. Eggs produced by aub+ females carrying an osk-bcd transgene synthesize Osk protein at both poles. In embryos produced by aub mutants carrying the osk-bcd transgene, Osk protein is absent from the posterior pole but efficiently translated at the anterior, reflecting a difference in the requirement for aub for translation of the transgenic and endogenous mRNAs (Wilson, 1996). However, while most of the Osk protein at the anterior pole is taken up in ectopic pole cells in embryos from aub heterozygous mothers, Osk protein particles become dispersed over the cortex in the anterior part of embryos from homozygous aub mutant mothers and fail to direct pole cell formation at the anterior pole. Thus, aub is required for the development of ectopic pole cells at the anterior of the embryo, and it is inferred that, like genes for other polar granule components, aub is involved in pole cell formation at the posterior as well (Harris, 2001).
Aub is found in particles that also contain Osk and are inferred to contain Vas, both known polar granule components. The Aub protein is located, like polar granules, at the posterior pole of the oocyte and embryo and are incorporated into pole cells. Although the posterior concentration of Aub in oocytes might seem to contribute to its role in translation of osk mRNA, which occurs predominantly at that site, several observations are at odds with this inference. In examination of more than 20 stage 8/9 egg chambers, it was discovered that the posterior accumulation of GFP-Aub is slightly delayed from the appearance of Osk protein in the same region. In the oocytes, significant Osk protein is often detected when very little GFP-Aub concentration at the posterior has occurred. In contrast, an egg chamber that had localized GFP-Aub but only low levels of Osk protein was never observed. Thus, it appears that Aub is not concentrated at the posterior before the onset of osk translation. No concentration of Aub is observed in the anterodorsal region of the oocyte where gurken (grk) mRNA is localized even though aub has also been implicated in the activation of grk translation (Harris, 2001).
Finally, it is possible to greatly reduce the posterior concentration of GFP-Aub and still initiate translation of Osk at normal levels. This situation occurs in vas mutants. Although previous reports have shown that vas mutant ovaries accumulate reduced levels of Osk protein, this study finds that Osk initially appears at normal levels in vas mutants. The previous analyses relied on Western blots of total ovarian protein, and so were unable to reveal any temporal specificity to the reduction. Confocal microscopy was used to compare Osk protein levels in ovaries and embryos from vas mutant and heterozygous females. Up to stage 11 of oogenesis, before deposition of the vitelline membrane interferes with whole-mount antibody staining, vas mutants accumulate normal amounts of Osk. By contrast, early embryos from vas mutant mothers display dramatic reductions in Osk protein. Thus, Osk protein levels are affected in vas mutants only in late stages of oogenesis, either through reduced translation or stability. The fact that normal levels of Osk appear in stage 9-11 vas mutant oocytes despite greatly lowered levels of GFP-Aub posterior accumulation, together with other observations, strongly suggest that the posterior concentration is not required for aub-dependent activation of osk translation (Harris, 2001).
Thus, the uniform low level of Aub found throughout the ooplasm appears to be sufficient for its action in osk and grk translation at the posterior pole and anterodorsal corner of the oocyte, respectively. The higher levels of Aub resulting from posterior recruitment presumably reflect other roles for Aub. These include two roles Aub shares with Vas: the posterior localization of nos mRNA (Wilson, 1996) and pole cell formation (Harris, 2001).
Drosophila Piwi-family proteins have been implicated in transposon control. This study examined piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein; Piwi and Aubergine were found to bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. These data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5' end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci (Brennecke, 2007).
Mobile genetic elements, or their remnants, populate the genomes of nearly every living organism. Potential negative effects of mobile elements on the fitness of their hosts necessitate the development of strategies for transposon control. This is critical in the germline, where transposon activity can create a substantial mutational burden that would accumulate with each passing generation. Hybrid dysgenesis exemplifies the deleterious effects of colonization of a host by an uncontrolled mobile element. The progeny of intercrosses between certain Drosophila strains reproducibly show high germline mutation rates with elevated frequencies of chromosomal abnormalities and partial or complete sterility (Bucheton, 1990; Castro, 2004; Kidwell, 1977). Studies of the molecular basis of this phenomenon (Pelisson, 1981; Rubin, 1982) linked the phenotype to transposon mobilization (Brennecke, 2007).
Hybrid dysgenesis occurs when a transposon, carried by a male that has established control over that element, is introduced into a naive female that does not carry the element. The transposon becomes active in the progeny of the naive female, causing a variety of abnormalities in reproductive tissues that ultimately result in sterility. Since the dysgenic phenotype is often not completely penetrant, a fraction of the progeny from affected females may survive to adulthood. Such animals can develop resistance to the mobilized element, although in many cases, several generations are required for resistance to become fully established (Pelisson, 1987). Immunity to transposons can only be passed through the female germline (Bregliano, 1980), indicating that there are both cytoplasmic and genetic components to inherited resistance (Brennecke, 2007).
Studies of hybrid dysgenesis have served a critical role in revealing mechanisms of transposon control. In general, two seemingly contradictory models have emerged. The first model correlates resistance with an increasing copy number of the mobile element. A second model suggests that discrete genomic loci encode transposon resistance. The first model is supported by studies of the I element. Crossing a male carrying full-length copies of the I element to a naive female leads to I mobilization and hybrid dysgenesis. The number of I copies builds during subsequent crosses of surviving female progeny until it reaches an average of 1015 per genome. At this point, I mobility is suppressed, as the initially nave strain gains control over this element. Thus, a gradual increase in I element copy number over multiple generations was implicated in the development of transposon resistance (Brennecke, 2007).
The second model, which attributes transposon resistance to specific genetic loci, is illustrated by studies of gypsy transposon control (Bucheton, 1995). Genetic mapping of gypsy resistance determinants led to a discrete locus in the pericentric b-heterochromatin of the X chromosome that was named flamenco (Pelisson, 1994). Females carrying a permissive flamenco allele (one that allows gypsy activity) showed a dysgenic phenotype when crossed to males carrying functional gypsy elements. Permissive flamenco alleles exist in natural Drosophila populations but can also be produced by insertional mutagenesis of animals carrying a restrictive flamenco allele. Despite extensive deletion mapping over the flamenco locus, no transposon repressor from flamenco has been identified. For P elements, a repressor of transposition has been identified as a 66 kDa version of the P element transposase. Expression of the repressor was proposed to correlate with increasing P element copy number, leading to a self-imposed limitation on P element mobility. However, studies of resistance determinants indicated that control over P elements could also be established by insertion of P elements into specific genomic loci, arguing for an alternative, copy number-independent control pathway. Studies of inbred lines or of wild isolates with natural P element resistance indicated that P insertions near the telomere of X (cytological position 1A) were sufficient to confer resistance if maternally inherited. Additionally, several groups isolated insertions of incomplete P elements in this same cytological location that acted as dominant transposition suppressors. Importantly, these defective P elements lacked sequences encoding the repressor fragment of transposase (Brennecke, 2007 and references therein).
Both models of transposon resistance, those determined by specific genomic loci and those caused by copy number-dependent responses might be linked to small RNA-based regulatory pathways. Copy number-dependent silencing of mobile elements is reminiscent of copy number-dependent transgene silencing in plants (cosuppression) and Drosophila. In both cases, silencing occurs through an RNAi-like response where high-copy transgenes provoke the generation of small RNAs, presumably through a double-stranded RNA intermediate. Moreover, mutations in RNAi pathway genes impact transposon mobility in flies and C.elegans. Finally, small RNAs (rasiRNAs) corresponding to transposons and repeats have been isolated from flies and zebrafish (Brennecke, 2007 and references therein).
At the core of the RNAi machinery are the Argonaute proteins, which directly bind to small RNAs and use these as guides for the identification and cleavage of their targets. In animals, Argonautes can be divided into two clades (Carmell, 2002). One contains the Argonautes, which act with microRNAs and siRNAs to mediate gene silencing. The second contains the Piwi proteins. Genetic studies have implicated Piwi proteins in germline integrity. For example, piwi mutations cause sterility and loss of germline stem cells. aubergine is a spindle-class gene that is required in the germline for the production of functional oocytes. The third Drosophila Piwi gene, Ago3, has yet to be studied. Mutation of Piwi-family genes also affects mobile elements. For example, piwi mutations mobilize gypsy, and aubergine mutations impact TART and P elements. Finally, both Piwi and Aubergine bind rasiRNAs targeting a number of mobile and repetitive elements. These complexes are enriched for antisense small RNAs, as might be expected if they were actively involved in silencing transposons by recognition of their RNA products (Brennecke, 2007 and references therein).
Recently, a new class of small RNAs, the piRNAs, was identified through association with Piwi proteins in mammalian testes. These 26-30 nt RNAs are produced from discrete loci, generally spanning 50-100 kb. Interestingly, mammalian piRNAs are relatively depleted of transposon sequences. Despite apparent differences in the content of Piwi-associated RNA populations in mammals and Drosophila, Piwi-family proteins share essential roles in gametogenesis, with all three murine family members, Miwi2 , being required for male fertility. In order to probe mechanisms of transposon control in Drosophila and to understand the relationship between Piwi protein function in flies and mammals, a detailed analysis was undertaken of small RNAs associated with Piwi proteins in the Drosophila female germline. These studies indicate that Drosophila Piwi-family members function in a transposon surveillance pathway that not only preserves a genetic memory of transposon exposure but also has the potential to adapt its response upon contact with active transposons (Brennecke, 2007).
In C. elegans, effective RNAi depends upon an amplification mechanism. Small RNAs from the primary dsRNA trigger are largely dedicated to promoting the use of complementary targets as templates for RNA-dependent RNA polymerases (RdRPs) in the generation of secondary siRNAs. In Drosophila, no RdRPs have been identified. However, the ability of Piwi-mediated cleavage to prompt the production of new piRNAs could create an amplification cycle that serves the same purpose as the RdRP-driven secondary siRNA generation systems in worms (Brennecke, 2007).
The cycle, termed Ping-Pong amplification (Aravin, 2007; see Properties and biogenesis of piRNA) is initiated by generating primary piRNAs, sampled from the piRNA clusters that have been identified in this study. As these are composed mainly of defective transposon copies, they serve as a genetic memory of transposons to which the population has been exposed. piRNAs that are antisense to expressed, dispersed transposons would identify and cleave their targets, resulting in the genesis of a new, sense piRNA in an Ago3 complex. The Ago3 bound sense piRNA would then seek a target, probably a precursor transcript from a master control locus that contains antisense transposon sequences. Ago3-directed cleavage would then generate additional antisense piRNAs capable both of actively silencing their target element and reinforcing the cycle through the creation of additional sense piRNAs (Brennecke, 2007).
The existence of such an amplification cycle essentially permits sense and antisense piRNAs act in concert to increase production of silencing-competent RNAs in response to the activity of individual transposons. Since Argonautes act catalytically, a significant amplification of the response could be achieved by even a relatively low level of sense piRNAs in Ago3 complexes. This model predicts that piRNAs participating in this process, namely those with complementary partners, should be more abundant than piRNAs without detectable partners. In accord with this hypothesis, the most frequently cloned Aub and Ago3-associated piRNAs show an increased probability of having partners within the data set. Interestingly, Piwi-associated RNAs do not follow this pattern. Since the amplification cycle consumes target transposon transcripts as part of its mechanism, posttranscriptional gene silencing may be sufficient to explain transposon repression. However, the possibility that transcriptional silencing may also be triggered by Piwi-family RNPs cannot be ruled out (Brennecke, 2007).
The amplification cycle may not be absolutely essential for silencing of all elements, since loci such as flamenco may operate in somatic follicle cells where the absence of Aub and Ago3 forces it to act in a stoichiometric fashion. In this regard, flamenco is unusual in that the vast majority of transposon fragments within this locus exist in a common orientation, which can lead to the production of antisense primary piRNAs given a long, unidirectional, precursor transcript (Brennecke, 2007).
In contrast to flamenco, most piRNA loci appear to be both bidirectionally transcribed and contain transposon sequences in random orientation. Nevertheless, the marked asymmetry of Piwi/Aub and Ago3 complexes is conserved in piRNAs that uniquely map to clusters. Among piRNAs that match transposons, 77% and 79% of unambiguously cluster-derived Piwi- and Aub-associated RNAs are antisense, while 73% of those in Ago3 are sense. These observations strongly suggest that piRNA clusters themselves participate in choice. According to this model, the remarkable strand asymmetry in piRNA populations hinges on informative interactions between master control loci and active transposons, which by their nature produce sense RNAs. These observations identify Ago3 as the principal recipient of piRNAs derived from transposon mRNAs. Thus, as long as there is an input to the system from active transposon transcripts via Ago3 and a preferential relationship between Ago3 and Aub for generating secondary piRNAs in their reciprocal complexes, a strand bias can be maintained even if primary and secondary piRNAs can both be derived from master control loci (Brennecke, 2007).
The amplification cycle must be initiated by primary piRNAs. Presently, the biogenesis pathway that generates primary piRNAs from piRNA clusters remains obscure. The data suggest that the piRNA precursor is a long, single-stranded transcript that is cleaved, preferentially at U residues. Transcripts have been detected from piRNA loci by RT-PCR that encompass multiple transposon fragments and find numerous small RNAs that cross junctions between adjacent transposons. In the case of flamenco, P element insertions near the proximal end of the locus have a polar effect both on these long RNA transcripts and on flamenco piRNAs (Brennecke, 2007).
Equally mysterious is the generation of piRNA 3' ends. Mature piRNAs could arise by two cleavage events and subsequent loading into Piwi complexes. Alternatively, piRNAs could be created following 5' end formation and incorporation of a long RNA into Piwi by resection of their 3' ends. The latter model is attractive, since it could provide an explanation for observed size differences between RNAs bound to individual Piwi proteins, since piRNA size would simply reflect the footprint of each Piwi protein. Although de novo biogenesis mechanisms must exist, maternally inherited piRNA complexes could also serve to initiate the amplification cycle. All three Piwi proteins are loaded into the developing oocyte, and Piwi and Aub are concentrated in the pole plasm, which will give rise to the germline of the next generation. Inherited piRNAs could enhance resistance to transposons that are an ongoing challenge to the organism, augmenting zygotic production of primary piRNAs. Indeed, maternally loaded rasiRNAs were detected in early embryos, and their presence was correlated with suppression of hybrid dysgenesis in D. virilis (Brennecke, 2007).
These data point to a comprehensive strategy for transposon repression in Drosophila that incorporates both a long-term genetic memory and an acute response to the presence of potentially active elements in the genome. The model that emerges from these studies shows many parallels to adaptive immune systems. The piRNA loci themselves encode a diversity of small RNA fragments that have the potential to recognize invading parasitic genetic elements. Throughout the evolution of Drosophila species, a record of transposon exposure may have been preserved by selection for transposition events into these master control loci, since this is one key mechanism through which control over a specific element can be achieved. Evidence has already emerged that X-TAS can act as a transposition hotspot for P elements, raising the possibility the piRNA clusters in general may attract transposons. Once an element enters a piRNA locus, it can act, in trans, to silencing remaining elements in the genome, either directly through primary piRNAs or by engaging in the amplification model described above. A comparison of D. melanogaster piRNAs to transposons present in related Drosophilids shows a lack of complementarity when comparisons are made at high stringency. However, when even a few mismatches are permitted, it is clear that piRNA loci might have some limited potential to protect against horizontal transmission of these heterologous elements. The existence of a feed-forward amplification loop can be compared to clonal expansion of immune cells with the appropriate specificity following antigen stimulation, leading to a robust and adaptable response (Brennecke, 2007).
In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. Small RNAs were sequenced associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI (Gunawardane, 2007).
Small noncoding RNAs trigger various forms of sequence specific gene silencing, including RNA interference (RNAi), translational repression, and heterochromatin formation in a variety of eukaryotic organisms, commonly referred to as RNA silencing. Members of the Argonaute family of proteins are essential components of RNA silencing. In Drosophila, five genes encode distinct members of the Argonaute family: AGO1, AGO2, Aubergine (Aub), Piwi, and AGO3. AGO1 and AGO2 constitute the Argonaute (AGO) subfamily and bind microRNA (miRNA) and small interfering RNA (siRNA), respectively. Aub, Piwi, and AGO3 belong to the PIWI subfamily of the Argonaute family and are enriched in germline cells (Williams, 2002), and Aub and Piwi have been shown to play important roles in germline cell formation. They are involved in silencing retrotransposons and other repetitive elements and exhibit target RNA cleavage (slicing) activity in vitro . Both Aub and Piwi associate with repeat-associated siRNAs (rasiRNAs) (Vagin, 2006; Saito, 2006). Aub- and Piwi-associated rasiRNAs are derived mainly from the antisense strand of retrotransposons, with little or no phasing, and have a strong preference for uracil (U) at the 5' end. Small RNA processing factors such as Dicer and Drosha are known to cleave preferentially at the 5' side of U; however, rasiRNAs are thought to be produced by a Dicer-independent pathway (Vagin, 2006). The mechanisms governing rasiRNA production remain to be elucidated (Gunawardane, 2007).
Very little is known about the function of AGO3, the third member of the Drosophila PIWI subfamily. Full-length cDNA of AGO3 revealed that the AGO3 gene is 83 kb in length. Peptide sequence alignments among Drosophila Argonaute proteins revealed that AGO3 is most similar to Piwi. The Asp-Asp-His motif in the PIWI domain, originally identified as the catalytic center for Slicer activity in human AGO2, is conserved in AGO3 (Gunawardane, 2007).
Embryonic RNA expression patterns of AGO3 are very similar to those of Piwi and Aub; they are expressed maternally, but their expression disappears by embryonic stages 10 to 12. To confirm these results, a monoclonal antibody (mAb) to AGO3 was produced, that revealed that AGO3 is strongly expressed in earlier embryonic stages but decreases as development proceeds. AGO3 accumulates in the cytoplasm of germline cells including germline stem cells (GSCs), germline cyst cells, nurse cells, and oocytes at earlier stages. In testes, AGO3 is expressed in GSC, primary gonial cells, and early spermatocytes. Unlike Piwi, AGO3 expression was undetected in the hub, a tiny cluster of postmitotic somatic cells localized at the apical tip of the testis that functions as a niche for GSC). Thus, with respect to expression in germline cells, AGO3 is more similar to Aub than to Piwi (Gunawardane, 2007).
All of the other members of the fly Argonautes are specifically associated with a subset of small RNAs: siRNAs, miRNAs, or rasiRNAs. Therefore whether AGO3 also associates with small RNAs produced in the fly ovary was tested. Immunoprecipitation with AGO3 mAb from ovary lysate revealed small RNAs 23 to 26 nucleotides (nt) long. The size distribution of AGO3-associated small RNAs is similar to that of Aub-associated small RNAs; in both cases, the peak is 24 nt and the longest is 27 nt. Small RNAs associated with AGO3 are likely to lack either a 2' or 3' hydroxyl group, because they do not migrate faster after beta-elimination as opposed to a synthetic siRNA that has 2' and 3' hydroxyl groups at the 3' end, the latter being the hallmarks of Dicer cleavage. These results suggest that AGO3-associated small RNAs in the ovary are produced by a pathway similar to those involved in production of rasiRNAs that associate with Aub and Piwi (Gunawardane, 2007).
A cDNA library was constructed of small RNAs associated with AGO3 in the ovary. Of 420 clones sequenced, 410 matched Drosophila genomic sequences in a database search, and most were rasiRNAs (86%; 353 of 410), as in the case of Aub and Piwi. Like rasiRNAs associated with Aub or Piwi, rasiRNAs associated with AGO3 included various kinds of transposable elements, both LTR (long terminal repeat) retrotransposons and LINE (long interspersed nuclear element)like elements. rasiRNAs associated with Aub or Piwi in ovaries are derived mainly from the antisense strand of retrotransposons, and the 5' end is predominantly U. These characteristics were not found for rasiRNAs associated with AGO3. However, AGO3-associated rasiRNAs were derived mainly from the sense strand of retrotransposons (82%), and they showed a strong preference for adenine (A) at nucleotide 10, but no preference for U at the 5' end. These results suggest that AGO3-associated rasiRNAs belong to a subset of rasiRNAs that are distinct from Aub- and Piwi-associated rasiRNAs (Gunawardane, 2007).
Some Argonaute proteins exhibit Slicer activity that directs cleavage of its cognate mRNA target across from nucleotides 10 and 11, measured from the 5' end of the small RNA guide strand. Thus, these findings suggest a model for rasiRNA biogenesis, in which the 5' end of Aub- and Piwi-associated rasiRNAs is determined and cleaved by AGO3-rasiRNA complexes, and the 5' end of AGO3-associated rasiRNAs is determined by Aub- and Piwi-rasiRNA complexes through a similar rasiRNA-guided cleavage event. For instance, AGO3 associated with a rasiRNA with A at nucleotide 10 can target a long RNA molecule by Watson-Crick base pairing and cleave the target RNA, resulting in sliced RNAs with U at the 5' end. Similarly, when Aub or Piwi associated with rasiRNAs with U at the 5' end slices its cognate RNA target, the resulting cleaved RNA will have A at nucleotide 10 (Gunawardane, 2007).
To test this model, AGO3 was examined for Slicer activity by performing in vitro target RNA cleavage assays with glutathione S-transferase (GST)AGO3 fusions. The target RNA, luc passenger siRNA (21 nt long, 5' end labeled with 32P), was efficiently cleaved by GST-AGO3, as was the case for GST-AGO1 and GST-Aub. The size of the cleaved products (9 nt) indicated that they direct cleavage of target RNA across from nucleotides 10 and 11 as measured from the 5' end of the small RNA guide strand. Both GST-Aub and GST-AGO3 with a longer guide RNA (26 nt) were also able to cleave a long transcript (180 nt). Long precursors of rasiRNAs both in sense and antisense orientations appear to exist in fly ovaries. These results corroborate the model in which the 5' end of rasiRNAs within the precursors is determined by rasiRNAs and cleaved by members of PIWI that associate with these rasiRNAs (Gunawardane, 2007).
The model predicts that some AGO3-associated rasiRNAs should be complementary to the first 10 nt of Aub- and Piwi-associated rasiRNAs. Sequence comparison between AGO3- and Aub-associated rasiRNAs indeed revealed pairs of rasiRNAs that show complementarities at their first 10 nt. Sixteen of 353 AGO3-associated rasiRNAs had such pairs with 11 of 676 Aub-associated rasiRNAs. However, such pairings were only found between AGO3- and Aub-associated rasiRNAs, and no pairs were observed between AGO3- and Piwi-associated rasiRNAs (353 versus 330). Like Aub-associated rasiRNAs, Piwi-associated rasiRNAs arise mainly from the antisense strand and their 5' ends show a strong preference for U; thus, it is difficult to argue that Piwi is not involved in this type of rasiRNA biogenesis. One possible reason is that Piwi is nuclear, whereas AGO3 and Aub are cytoplasmic. This type of rasiRNA biogenesis may operate in the cytoplasm. Alternatively, formation of 5' ends of Piwi-associated rasiRNAs may occur only at an earlier time during germline development (Gunawardane, 2007).
RasiRNAs are involved in genome surveillance by silencing repetitive elements and controlling their mobilization in the Drosophila germ line. It was recently shown that rasiRNAs are produced by a mechanism that requires neither Dicer-1 nor Dicer-2 in flies. These data suggest that rasiRNAs in a sense orientation guide formation of the 5' end of rasiRNAs in an antisense orientation, and vice versa; as well, this cycle of mutual dependency elaborates optimal rasiRNA production. In this model, proteins of the PIWI subfamily function as Slicer for formation of the 5' end during rasiRNA biogenesis. This model requires that sliced rasiRNA precursors then be cleaved again at the 3' end by an as yet unidentified endonuclease (or nibbled by exonuclease) to produce mature rasiRNAs before or after loading of the resulting cleavage products onto another member of the PIWI. Once 'primary' complexes of rasiRNAs with proteins of PIWI are produced, these complexes will in turn function as the 'initiator' of secondary rasiRNA biogenesis, and so nascent rasiRNAs should be continuously supplied in the ovary and testis. Such a process may occur through rasiRNA germline transmission. Of the PIWI members, at least Aub is accumulated to the posterior pole in oocytes and remains in polar granules in early embryos. It is then incorporated in pole cells, the progenitor of the Drosophila germ line (10) (Gunawardane, 2007).
Piwi-interacting RNAs (piRNAs) consist of a germline-specific group of small RNAs derived from distinct intergenic loci in the genome. piRNAs function in silencing selfish transposable elements through binding with the PIWI subfamily proteins of Argonautes. This study shows that piRNAs in Drosophila are 2'-O-methylated at their 3' ends. Loss of Pimet/Hen1 (piRNA methyltransferase), the Drosophila homolog of Arabidopsis HEN1 methyltransferase for microRNAs (miRNAs), results in loss of 2'-O-methylation of fly piRNAs. Recombinant Pimet shows single-stranded small RNA methylation activity in vitro and interacts with the PIWI proteins within Pimet mutant ovary. These results show that Pimet mediates piRNA 2'-O-methylation in Drosophila (Saito, 2007; full text of article).
In Pimet mutant ovary, piRNAs associated with Aub and Piwi were not methylated at the 3' ends, most likely due to loss of Pimet expression. Whether GST-Pimet is able to methylate these piRNAs associated with the PIWI proteins from Pimet mutant ovary was investigated. Aub-piRNA complexes were immunopurified with a specific antibody against Aub and subjected to in vitro methylation assays. As a control, miRNAs associated with AGO1 were also obtained through immunoprecipitation using anti-AGO1 from ovary lysate. It was found that piRNAs were methylated even in a complex form with Aub. piRNA methylated in the assay showed resistance to oxidation and β-elimination treatment. Interestingly, miRNAs associated with AGO1 were not methylated, although these miRNAs are single-stranded in a complex form with AGO1. Confirmation that the miRNA levels were several-fold higher than those of piRNAs was provided by phosphorylation of these small RNAs. It seems that small RNA methylation by Pimet is largely influenced by the accessibility of the 3' ends of the substrates to Pimet itself. Structural analysis of Argonaute proteins suggests that the 5' end of the small guide RNA is anchored in a highly conserved pocket in the PIWI domain, whereas the 3' end of the small RNA is embedded in the PAZ domain. Taken together, these results suggest that the 3' ends of Aub-associated piRNAs are not tightly bound to the PAZ domain, but are exposed to the surface of the protein. In contrast, the 3' ends of AGO1-associated miRNAs are likely to be embedded in the PAZ domain and therefore are not exposed to the surface of the protein. Alternatively, but not mutually exclusively, it is conceivable that Pimet may interact only with PIWI proteins and not with AGO proteins, thereby methylating only small RNAs associated with PIWI proteins. To test this, whether Pimet associates with PIWI proteins was investigated. A GST pull-down assay was performed; GST-Pimet was first incubated with Pimet mutant ovary lysate, and after extensive washing the eluates were probed with PIWI protein antibodies. Aub, Piwi, and AGO3 were clearly detected in the bound fraction with GST-Pimet but not with GST itself. By contrast, AGO1 was not observed. These results indicated that Pimet is capable of physically interacting with PIWI proteins containing piRNAs that can serve as substrates for Pimet methylation. Addition of RNaseA did not affect the interaction of Pimet with Aub, suggesting that Pimet is able to associate directly with the PIWI proteins. In Drosophila, piRNA methylation may occur after matured piRNAs are loaded onto PIWI proteins. If so, it clearly differs from the case of miRNA methylation in plants, which likely occurs prior to miRNA loading onto the AGO proteins when miRNAs are still in a duplex form with the complementary miRNA* molecules (Saito, 2007).
Mutations in Arabidopsis hen1 cause reduced fertility. Thus, is the piRNA methylation by Pimet crucial in Drosophila? piRNAs function in genome surveillance in germlines in concert with PIWI proteins. Mutations in aub, piwi, and others like spindle-E (homeless) cause piRNAs not to be accumulated in gonads, and lead to germ cell malformation and sterility. This clearly indicates that piRNAs are necessary for perpetuation of organisms. However, the Pimet mutant fly seems to be viable and fertile. Steady-state levels of piRNAs in the methylation-defective mutant are also similar to those in wild type. Expression levels of retrotransposons do not seem to be changed by loss of Pimet expression. Thus, the function of 3' end methylation is currently unknown. Further investigation such as by immunohistochemistry may be required to obtain a more detailed morphology of the mutant. Extensive analyses of the mechanisms underlying piRNA methylation may also provide important clues to more fully elucidating piRNA biogenesis. Aub and AGO3, which determine and form the 5' end of piRNAs in piRNA biogenesis, were shown to be in the protein fraction associated with Pimet. Identifying more Pimet-associated proteins may reveal the factors required for formation of the 3' end of piRNAs (Saito, 2007).
The study of P-element repression in Drosophila led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. This study reports that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3-7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway (Josse, 2007; full text of article).
Repression of transposable elements (TEs) involves complex mechanisms that can be linked to either small RNA silencing pathways or chromatin structure modifications depending on the species and/or the TE family. Drosophila species are particularly relevant to the study of these repression mechanisms since some families of TEs are recent invaders, allowing genetic analysis to be carried out on strains with or without these TEs. In some cases, crossing these two types of strains induces hybrid dysgenesis, a syndrome of genetic abnormalities resulting from TE mobility. In D. virilis, repression of hybrid dysgenesis has been correlated to RNA silencing since small RNAs of the retroelement Penelope, responsible for dysgenesis, were detected in nondysgenic embryos but not in dysgenic embryos. In D. melanogaster, repression of retrotransposons can be established by noncoding fragments of the corresponding element (I factor, ZAM, and Idefix) and can be in some cases (gypsy, mdg1, copia, Het-A, TART, and ZAM, Idefix) sensitive to mutations in genes from the Argonaute family involved in small RNA silencing pathways. In the same species, strong repression of the DNA P TE, by a cellular state that has been called 'P cytotype', can be established by one or two telomeric P elements inserted in heterochromatic 'Telomeric Associated Sequences' (TAS) at the 1A cytological site corresponding to the left end of the X chromosome. This includes repression of dysgenic sterility resulting from P transposition. This P cytotype is sensitive to mutations affecting both Heterochromatin Protein 1 (HP1) (Ronsseray, 1996) and the Argonaute family member AUBERGINE (Reiss, 2004). P repression corresponds to a new picture of TE repression shown, using an assay directly linked to transposition, to be affected by heterochromatin and small RNA silencing mutants (Josse, 2007).
In the course of the study of P cytotype, a new silencing phenomenon has been discovered. Indeed, a P-lacZ transgene or a single defective P element inserted in TAS can repress expression of euchromatic P-lacZ insertions in the female germline in trans, if a certain length of homology exists between telomeric and euchromatic insertions. This homology-dependent silencing phenomenon has been termed Trans-Silencing Effect (TSE) (Roche, 1998). Telomeric transgenes, but not centromeric transgenes, can be silencers and all euchromatic P-lacZ insertions tested can be targets. TSE is restricted to the female germline and has a maternal effect since repression occurs only when the telomeric transgene is maternally inherited (Ronsseray, 2001). Further, when TSE is not complete, variegating germline lacZ repression is observed from one egg chamber to another, suggesting a chromatin-based mechanism of repression. Recently, an extensive analysis of small RNAs complexed with PIWI family proteins (AUBERGINE, PIWI, and AGO3) was performed in the Drosophila female germline. The latter study showed that most of the RNA sequences associated to these proteins derive from TEs. TSE corresponds likely to such a situation (Josse, 2007).
This study analyzed the genetic properties of TSE and shows that it has an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted stimulating component. Further, in order to investigate the mechanism behind TSE, a candidate gene analysis was performed to identify genes whose mutations impair TSE. It was found that TSE is strongly affected both by mutations in genes involved in heterochromatin formation and in the recently discovered small RNA silencing pathway called 'repeat-associated small interfering RNAs' (rasiRNA) pathway. In contrast, this study shows that TSE is not sensitive to genes specific to the classical RNA interference pathway linked to small interfering RNAs (siRNA) or to the micro RNA (miRNA) pathway. This suggests thus that TSE involves a rasiRNA pathway linked to heterochromatin formation and that such a mechanism, working in the germline, may underlie epigenetic transmission of repression through meiosis (Josse, 2007).
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