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) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, 'piRNA clusters.' piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. This study describes strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. It was found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. A second piRNA pathway was found centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line (Li, 2009).
The ability to tame transposons while retaining them in the genome is a particular specialty of eukaryotes. Transposons, repetitive sequences, and other forms of 'selfish' DNA comprise as much as 42% of the human genome and nearly 30% of the genome of Drosophila melanogaster. In metazoa, transposons are silenced by the piRNA pathway, which is guided by 23-30 nt RNAs. The piRNA pathway is distinct from other RNA silencing pathways in that its small RNA guides are not produced by dicing. In contrast, both small interfering RNAs (siRNAs) and microRNAs (miRNAs) are cleaved by double-stranded RNA-specific endonucleases, Dicers, to yield double-stranded intermediates-siRNA or miRNA/miRNA* duplexes-that are loaded into members of the Argonaute family of proteins. piRNAs, too, act as guides for Argonaute proteins, but they appear not to exist as stable double-stranded intermediates at any point in their biogenesis. piRNAs bind PIWI proteins, a sub-family of Argonaute proteins that are expressed in germ-line cells. PIWI proteins were first identified by their roles in maintaining and patterning Drosophila germ cells. The defects in the organization of embryonic pattern in piRNA pathway mutations are likely an indirect consequence of their larger role in maintaining genomic stability. The three Drosophila PIWI proteins, Piwi, Aubergine (Aub), and Argonaute3 (Ago3), are expressed in the male and female germ line cells (Li, 2009).
The prevailing model for piRNA biogenesis-the 'ping-pong' model-reflects the discovery that the first 10 nt of piRNAs bound to Aub or Piwi, which are largely antisense and typically begin with uridine, are often complementary to the first 10 nt of piRNAs bound to Ago3, which are largely sense and typically bear an adenosine at position 10. Many Argonaute proteins can act as RNA-guided RNA endonucleases, and all such Argonautes cut their target RNAs 5' to the base that pairs with the tenth nucleotide of the small RNA guide; all three fly PIWI proteins retain their endonuclease activity. Thus, the observed 10 nt 5' complementarity between piRNAs suggests that the 5' ends of piRNAs bound to Aub or Piwi are defined by Ago3-catalyzed cleavage, and, reciprocally, that the 5' ends of piRNAs bound to Ago3 are defined by piRNAs bound to Aub or Piwi. The ping-pong model seeks to explain these observations, as well as the role of piRNA cluster transcripts in piRNA biogenesis, the function of piRNAs in silencing transposons, and the extraordinary antisense bias of piRNAs generally. At its core, the model proposes that piRNAs participate in an amplification loop in which transposon sense transcripts (e.g., transposon mRNAs) trigger the production of new, antisense piRNAs. Ago3, guided by sense piRNAs, lies at the heart of the amplification loop (Li, 2009).
To test for the ping-pong model, strong loss-of-function mutations were isolated in ago3. This study report the detailed analysis of ago3 and aub mutant flies. The data provides strong support for an amplification cycle in which Ago3 amplifies piRNA pools and enforces on them a strong antisense bias, increasing the number of piRNAs that can act to destroy transposon mRNAs. Moreover, a second, perhaps somatic, piRNA pathway was detected, centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Most of the transposons targeted by this second pathway reside in the flamenco piRNA cluster, which was first identified as a repressor of transposon expression in somatic follicle cells (Li, 2009).
Because ovaries contain both germ-line and somatic cells, the data conflate two distinct cell lineages. Combining the data with extensive genetic studies of gypsy and other transposon families represented in the flamenco locus, this study has attempted to disentangle germ-line and somatic piRNA function. It is proposed that the somatic piRNA pathway is the more straightforward, involving only Piwi and not Ago3 or Aub. The data suggest that Piwi cannot act alone to amplify piRNAs. It is envisioned that Piwi-bound piRNAs in the soma are produced by a ribonuclease that randomly generates single-stranded guides that are subsequently loaded into Piwi and trimmed to length. Although Piwi-bound piRNAs generally begin with U and Piwi shows in vitro a preference for binding small RNA that begins with U, current evidence cannot distinguish between a putative piRNA-generating ribonuclease cleaving mainly at U and Piwi selecting U1 piRNAs from a set of RNAs with all possible 5' nucleotides (Li, 2009).
A model is presented for piRNA biogenesis. The Aub- and Ago3-dependent piRNA amplification cycle is envisioned to operate only in the germ line, whereas a Piwi-dependent, Aub- and Ago3-independent pathway is shown for somatic cells. In the germ line, Piwi can also partner with Ago3 to amplify piRNAs. Without an amplification cycle to ensure an antisense bias, some other mechanism must operate to explain why Piwi-bound piRNAs are overwhelmingly antisense. A plausible but somewhat unsatisfying explanation comes from flamenco itself, whose constituent transposons are nearly all oriented in a single direction, so that the ∼160 kb flamenco transcript is almost entirely antisense to the transposons. How such a non-random array of transposons could arise is unknown. Other non-randomly oriented piRNA clusters may explain the smaller number of transposons in group III that are not present in flamenco (Li, 2009).
The transposons in most piRNA clusters do not show such a pronounced non-random orientation. These likely act in the germ line to produce primary piRNAs that load into Aub. The observed antisense bias of Aub-bound piRNAs arises subsequently, when Aub generates Ago3-bound secondary piRNAs and Ago3 acts, in turn, to produce Aub-bound secondary piRNAs. It is proposed that in the absence of Ago3, the sense/antisense ratio of Aub-bound piRNAs reverts to the inherent sense/antisense bias of the transposable element sequences present in the transcripts of piRNA clusters (Li, 2009).
For this cycle to skew the Aub-bound piRNA population toward antisense, the substrate for cleavage by primary piRNA-bound Aub must be largely sense RNA. The best candidate for such sense RNA is mRNA derived from actively transcribed transposon copies. If such sense mRNA were largely found in the cytoplasm, it would be spatially segregated from the cluster transcripts, which is envisioned to be retained in the nucleus. Supporting this idea, sense transcripts from the group I transposon, I element, normally accumulate only in the nuclei of germ-line nurse cells (Chambeyron, 2008), likely because they are destroyed in the cytoplasm by Aub-bound primary piRNAs and Aub-bound piRNAs produced by Ago3-dependent amplification. In the absence of Aub, these sense transposon transcripts accumulate in the cytoplasm instead, consistent with the strong desilencing of I element in aub and ago3 mutants (Li, 2009).
The piRNA ping-pong hypothesis predicts a role for Ago3 in the production of Aub-bound antisense piRNAs, but the current finding that loss of Ago3 also reduced the abundance of Piwi-bound antisense piRNAs was unexpected. The majority of Aub and Ago3 is found in nuage and in the cytoplasm, but Piwi is predominantly nuclear. How then can Ago3 direct the production of Piwi-bound piRNAs? Perhaps Piwi transits the nuage en route from its site of synthesis, the cytoplasm, to where it accumulates, the nucleus. In this view, cytoplasmic Piwi is predicted to lack a small RNA guide. Piwi would then acquire its small RNA guide in the nuage, through a process that requires Ago3. Loading a piRNA into Piwi might then license it for entry into the nucleus, where it could act post-transcriptionally or transcriptionally to silence transposon expression. In this view, mutations in genes required for nuage assembly or stability, such as vasa, as well as genes required for Piwi loading would reduce the amount of nuclear Piwi. A similar mechanism may operate in mammals, where the PIWI protein MILI is found in cytoplasmic granules, whereas MIWI2 is nuclear. In the absence of MILI, MIWI2 delocalizes from the nucleus to the cytoplasm, although MIWI2 is not required for the localization of MILI (Li, 2009).
Such a model cannot explain the loading of Piwi in the somatic follicle cells, which contain little or no Ago3 or Aub and which do not contain nuage. A simple but untested hypothesis for these cells is that in the absence of nuage, empty Piwi readily enters the nucleus, where it obtains its small RNA guide. It might reasonably be expected that in germ cells the absence of nuage would impair the loading of Piwi by eliminating the Ago3-dependent, germ-line specific Piwi-loading process, but also facilitate entry of some empty Piwi into the nucleus, where it could obtain small RNA guides. Consistent with this idea, some Piwi is detected in the nucleus in ago3 ovaries. The simplicity of this hypothesis, of course, belies the complexity of testing it (Li, 2009).
Why are there two distinct piRNA production pathways? Retrotransposons 'reproduce' by producing sense RNA encoding transposases and other proteins that allow them to jump to new locations in the germ cell genome. The conservation of the piRNA ping-pong cycle in animals suggests that it is an ancient and conserved germ-line defense against retrotransposition. In flies, the gypsy family of retroelements appears to have moved its reproductive cycle to the somatic follicle cells adjacent to the germ line, which it infects using retrovirus-like particles. gypsy thus appears to avoid germ-line piRNA surveillance by transcribing and packaging its RNA in the soma. Perhaps expression of Piwi in Drosophila follicle cells reflects an adaptive evolutionary counter move to the gypsy reproductive strategy. The simplicity of the direct loading of Piwi with antisense piRNAs derived from flamenco may have made this counter defense more evolutionarily accessible than a strategy requiring expression of all the proteins needed for the Ago3:Aub ping-pong mechanism. In the future, more extensive analysis of the cellular and genetic requirements for ping-pong-independent and ping-pong-dependent piRNA mechanisms in Drosophila melanogaster and in more ancient animal species may provide a test for these ideas (Li, 2009).
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).
Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). This study reports that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). Piwi proteins are expressed in Xenopus oocytes and numerous Xenopus piRNAs were identified. This paper reports that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, capsuleen/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. These studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. These findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification (Kirino, 2009).
Piwi family proteins are expressed in the germline and bind ~26 to ~30 nucleotide (nt) piRNAs. Drosophila express three Piwi proteins: Aub, Piwi and Ago3. Mice also express three Piwi proteins termed Miwi, Mili/PiwiL2 and Miwi2/PiwiL4. Tens of thousands of distinct piRNAs have been described and most of them are species-specific. In Drosophila , Piwi proteins and piRNAs (also known as rasiRNAs - repeat associated small interfering RNAs) silence transposons in the germline. A similar function has been found for a subset of mouse and zebrafish piRNAs. An amplification loop of piRNAs has been described but how primary piRNAs are generated is unknown. sDMA modifications occur in sequence motifs composed of arginines flanked by glycines (GRG) or alanines (GRA or ARG) that are often found as repeats. PRMT5 and its cofactors MEP50/WD45 and pICln form the methylosome that methylates Sm proteins. A highly specific monoclonal antibody (17.8) was produced that recognizes Mili by Western blot, immunoprecipitation and immunofluorescence microscopy. By serendipity it was discovered that the widely used Y12 monoclonal antibody recognizes mouse Mili and Miwi proteins and their bound piRNAs. The Sm proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs) constitute the main antigen for Y12. piRNAs were not identified in immunoprecipitates of snRNPs, heterogeneous ribonucleoproteins (hnRNPs) or of the Survival of Motor Neurons (SMN) complex using various antibodies. By Northern blot analysis it was found that piR-1, but not miR-16, an abundant miRNA, was found in Y12 immunoprecipitates, suggesting that Y12 recognizes Piwi but not Ago proteins (Kirino, 2009).
The epitope that Y12 recognizes on Sm proteins consists of symmetrically dimethylated arginines, in the glycine-arginine rich regions of the proteins. It was reasoned that Y12 likely reacted with sDMA-containing epitopes in Mili and Miwi, and arginine residues were sought that could be symmetrically methylated. Intriguingly, it was found that most animal Piwi proteins contain sDMA motifs that are typically clustered close to the amino terminus, while no animal Ago proteins contained such motifs. However, it was found that four of ten Arabidopsis Ago proteins contained sDMA motifs (Kirino, 2009).
To test whether Miwi and Mili contain sDMAs, SYM11 and ASYM24 antibodies, which specifically recognize proteins containing sDMA-glycine or aDMA-glycine repeats, respectively, were used. SYM11, as well as Y12, reacted strongly with endogenous Miwi and Mili, while ASYM24 showed only faint reactivity towards endogenous Miwi. In contrast, recombinant Flag-Mili or Flag-Miwi purified from baculovirus-infected Sf9 cells, were not recognized by Y12 or SYM11 (or ASYM24). This is entirely consistent with the finding that recombinant human Sm proteins expressed in Sf9 cells also do not contain sDMAs because Sf9 cells do not express type II PRMTs and thus cannot produce sDMA modifications. These findings indicate that Mili and Miwi proteins contain sDMAs. The putative sDMA motifs of Miwi are concentrated very close to the amino terminus with the exception of one GRG triplet. Flag-tagged full-length Miwi and two truncated forms of Miwi (aa 68-862 or 1-212) were transfected in 293T cells, by Flag immunoprecipitation and subject to western blot with SYM11 antibody, SYM11 antibody recognizes the amino terminus of Miwi protein (Kirino, 2009).
Next it was asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. It was reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase three Xenopus Piwi proteins were identified which were named Xili, Xiwi and Xiwi2. All three Xenopus Piwi proteins contain putative sDMA motifs. Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ~95 kDa and ~110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis that were identified by mass spectrometry as Xiwi and Xili respectively. As shown in the western blots, Y12 recognizes both Xiwi and Xili, while anti-Mili (17.8) reacts only with Xili. In addition, both Xiwi and Xili are recognized by SYM11, indicating that Xiwi and Xili contain sDMAs (Kirino, 2009).
X. laevis piRNAs were isolated and analyzed from Y12 immunoprecipitates. ~26-29 nt piRNAs are present in the Y12 immunoprecipitates and their 3'-termini are not eliminated by periodate oxidation and are thus likely 2'-O-methylated, as seen in piRNAs from other species (Kirino, 2009).
Deep sequencing was performed of X. laevis piRNAs from Y12 immunoprecipitates of oocytes and testis. The nucleotide composition of X. laevis piRNAs shows enrichment of Uridine in the first nucleotide position and of Adenine in the tenth nucleotide position. There is also enrichment for piRNAs whose first 10 nucleotides are complementary to the first 10 nucleotide of other piRNAs. These features indicate that a fraction of X. laevis piRNAs target transposon transcripts and that they also participate in a piRNA amplification loop, as has been described for Drosophila and zebrafish piRNAs and prepachytene mouse piRNAs. By Northern blot XL-piR-3, a representative piRNA, is expressed specifically in oocytes and by in situ hybridization XL-piR-3 is localized predominantly in the cytoplasm of X. laevis oocytes and it is expressed in higher levels in immature oocytes (Kirino, 2009).
Genetic disruption of either Drosophila PRMT5 (dPRMT5; also know as capsuleen - csul- , and dart5) or its cofactor valois, (the Drosophila homolog of MEP50/WD45), results in complete loss of sDMA modifications of Sm proteins in ovaries. However, unlike the situation in mammals, the levels or function of Sm proteins is not affected by loss of sDMAs (Kirino, 2009).
Null or hypomorphic alleles of dPRMT5 (csul, dart5) phenocopy aub null alleles and it was reasoned that dPRMT5 might be the methyltransferase that produces sDMAs in Aub, Piwi and Ago3, in vivo. To test this, ovaries were used from csulRM50/Df(2R)Jp7 females, which give rise to embryos that are genetic nulls for dPRMT5 and w- was used as a wild-type control. Western blots of ovary lysates from wt and maternal null csul showed that there was near complete loss of SYM11 reactivity, indicating dramatic reduction of sDMA modified proteins in csul ovaries. There was no change in ASYM24 reactivity between wt and csul, indicating that aDMA modified proteins were not affected. These findings confirm that dPRMT5 (csul, dart5) activity is required specifically for sDMA modification. Piwi and Aub proteins were immunoprecipitated from wt and csul mutant ovaries and the immunoprecipitates were probed with SYM11 and ASYM24. SYM11 reacted very strongly with Aub and also with Piwi immunopurifed from wt but not csul ovaries; ASYM24 reacted only weakly with Aub from wt ovaries. Immunoprecipitates of Ago3 were also with SYM11 and ASYM24 and it was observed that only Ago3 from wt ovaries reacted with SYM11. These results indicate that, like the mouse and X. laevis Piwi family proteins, Drosophila Piwi, Aub and Ago3 contain sDMAs and that dPRMT5 is the methylase that produces sDMAs of these proteins (Kirino, 2009).
In Aub the four arginines that are putative substrates for symmetrical dimethylation are found in tandem very close to the amino terminus. Site-directed mutagenesis was used to change these arginines into lysines that are not subjected to methylation by PRMTs. Flag-tagged wild-type (WT) or mutant (M) Aub were stably transfected into Drosophila S2 cells (which express dPRMT5), the proteins were purified by Flag immunoprecipitation and subjected to western blot with Flag, SYM11 and ASYM24 antibodies. SYM11 antibody reacted only with wild-type Aub. Next the binding of wild-type and mutant Aub to a synthetic piRNA was assayed. Immunopurified, wild-type or mutant Flag-Aub were incubated with a 5'-end radiolabeled synthetic piRNA containing 4-thio-Uridine at the first position, followed by crosslinking with Ultraviolet light and NuPAGE analysis. There was similar binding between wild-type and mutant Aub proteins. These findings indicate that one or more of the four arginines in the amino terminus of Aub are symmetrically dimethylated and arginine methylation does not impact piRNA binding (Kirino, 2009).
Next, RNAs bound to Piwi and Aub were isolated and analyzed from wt or csul ovaries. piRNAs remain bound to Piwi and Aub proteins in the csul ovaries. There is mild reduction of Piwi-piRNAs and marked reduction of Aub-piRNAs in csul ovaries corresponding to concordant reduction of protein levels of Piwi and Aub. The Piwi associated piRNAs were gel purified and subjected to periodate oxidation followed by β-elimination and it was revealed that Piwi-associated piRNAs purified from csul ovaries retain 2'-O-methylation of their 3' termini. These findings indicate that the lack of sDMA modifications of Piwi and Aub in csul ovaries does not impair the methylation of piRNAs or their binding to Piwi and Aub (Kirino, 2009).
Next the protein levels of Piwi family proteins were compared between wt, heterozygous and homozygous csul ovaries. Western blot analysis showed that there was marked reduction of Aub and Ago3 protein levels and lesser reduction of Piwi levels in csul ovaries, whereas the levels of the miRNA binding protein Ago1 were not affected. Since mRNA levels of Aub, Piwi and Ago3 are the same between wt and csul ovaries, dPRMT5 activity might be required to stabilize the Aub, Ago3 and Piwi proteins most likely by symmetrically methylating their arginines. The level of a representative piRNA (roo-rasiRNA), was decreased in csul ovaries in accord with reduction of Piwi family proteins, while the level of a representative miRNA, miR-8, was not affected. The homozygous csul ovaries showed a 30-fold increase in the levels of the HeT-A retrotransposon transcript, whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline. Collectively these results indicate that loss of dPRMT5 activity impairs the amounts of Piwi proteins and piRNAs, resulting in disruption of their function of transposon silencing (Kirino, 2009).
Next the localization of Ago3, Aub and Piwi was analyzed by confocal microscopy in wt and homozygous csul early stage egg chambers. Previous studies have shown that Piwi is localized predominantly in the nuclei of follicle and germ cells while Ago3 and Aub are localized in the cytoplasm of germ cells. In oocytes, Aub is concentrated in the germ (pole) plasm. Representative images reveal that the level of Ago3 is markedly reduced in csul early stage egg chambers, while there is only a mild reduction of Aub and Piwi protein levels (Kirino, 2009).
Germ cell (PGC) formation in Drosophila requires that cytoplasmic determinants are localized to the posterior pole of the embryo. Genetic screens have identified grandchildless maternal genes that are required for PGC specification and invariably the protein or RNA products of these genes are concentrated in the pole plasm and are incorporated into the PGCs. Among these genes are Aub, dPRMT5 (csul, dart5), and its cofactor valois and tudor, whose protein product contains eleven tudor domains. The localization of Aub was tested in csul oocytes by confocal microscopy. Representative results show that the levels of Aub in the pole plasm of stage 10 egg chambers are markedly reduced. Western blotting reveals marked reduction of Aub protein levels in csul ovaries while confocal microscopy shows that Aub levels are subtly reduced in early stage egg chambers but markedly reduced in later stage egg chambers, suggesting that lack of sDMAs affects Aub levels at later stages in oogenesis (Kirino, 2009).
These studies show that sDMA modification of Piwi family proteins is a conserved post-translational modification, and the methyltransferase PRMT5 (csul/dart5) is identified as the enzyme that catalyzes sDMAs of Piwi, Ago3 and Aub in Drosophila ovaries, in vivo. Both Aub and csul/dart5 (dPRMT5) are grandchildless genes and the finding that Aub is a substrate for dPRMT5, indicates that an important function of dPRMT5 in pole plasm function and PGC specification involves methylation of Aub. Intriguingly, tudor domains bind to sDMAs and Tudor protein is also a grandchildless gene that is required for pole plasm assembly and function. These findings suggest that pole plasm function may involve an interacting network of genes whose protein products contain sDMAs (Aub), the methylase (dPRMT5) and its cofactor (valois/dMEP50) that produce sDMAs and tudor domain (Tudor) proteins that may bind to sDMA-containing proteins. It is noted that both PRMT5 and tudor-domain-containing genes are found in all species that express Piwi family proteins and knockout of tudor domain containing 1/mouse tudor repeat 1 in mice leads to spermatogonial cell death and male sterility. Furthermore, it is noted that other Drosophila proteins whose genes are required for piRNA accumulation or function, such as Spindle-E/homeless, contain tudor domains. It will be interesting to test whether tudor domain containing proteins interact with sDMA-modified Piwi family proteins and to elucidate their function (Kirino, 2009).
Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins, have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation and transcriptional and post-transcriptional silencing. The piRNA pathway has other essential functions in germline stem cell maintenance and in maintaining germline DNA integrity. This study uncovered an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo at the maternal-to-zygotic transition. In Drosophila, maternal mRNA degradation depends on the RNA-binding protein Smaug and the deadenylase CCR4, as well as the zygotic expression of a microRNA cluster. Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, it was found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in the nos 3' untranslated region. Reduced deadenylation when piRNA-induced regulation is impaired correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3' untranslated region, in the bulk of the embryo. It is proposed that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression (Rouget, 2010).
In Drosophila embryos, Nos is expressed as a gradient that emanates from the posterior pole and organizes abdominal segmentation. The majority of nos mRNA is distributed throughout the bulk cytoplasm, translationally repressed and subsequently degraded during the first 2-3 h of development. This repression is essential for head and thorax segmentation. A small amount of nos transcripts, localized at the posterior pole of the embryo, escapes degradation and is actively translated, giving rise to the Nos protein gradient. nos mRNA decay in the bulk cytoplasm depends on the CCR4-NOT deadenylation complex and its recruitment onto nos by Smaug (Smg). This contributes to translational repression in the bulk of the embryo and is required for embryonic antero-posterior patterning (Zaessinger, 2006; Rouget, 2010 and references therein).
Smg has been suggested to be not the only activator of nos mRNA decay during early embryogenesis. Zygotically expressed miRNAs have been reported to activate maternal mRNA deadenylation in zebrafish embryos and decay in Drosophila embryos. This study investigated the potential involvement of other classes of small RNAs in mRNA deadenylation and decay before zygotic expression. Because piRNAs are expressed maternally in the germ line and are present in early embryos, the possible role of the piRNA pathway in maternal mRNA deadenylation was analyzed. Piwi, Aubergine (Aub) and Ago3 are specific Argonaute proteins, Armitage (Armi) and Spindle-E (Spn-E) are RNA helicases, and Squash (Squ) is a nuclease involved in piRNA biogenesis and function. Poly(A) test assays were performed to measure nos mRNA poly(A) tail length in embryos spanning 1-h intervals during the first 4 h of embryogenesis. In contrast to the progressive shortening of nos mRNA poly(A) tails observed in wild-type embryos correlating with mRNA decay during this period, nos poly(A) tail shortening was affected in embryos from females mutant for the piRNA pathway (herein referred to as mutant embryos). This defect in deadenylation correlated with higher amounts of nos mRNA in mutant embryos, as quantified by reverse transcription-quantitative PCR (RT-qPCR). In situ hybridization revealed stabilized nos mRNA in the bulk cytoplasm of mutant embryos where it is normally degraded in the wild type. Consistent with previous data showing that nos mRNA deadenylation is required for translational repression (Zaessinger, 2006), defective deadenylation in mutant embryos resulted in the presence of ectopic Nos protein throughout the embryo. The presence of Nos in the anterior region results in the repression of bicoid and hunchback mRNA translation and in affected head skeleton. It was found that the piwi1 mutant embryos that were able to produce a cuticle had head defects (Rouget, 2010).
The piRNA pathway has a role during early oogenesis in preventing DNA damage, possibly through the repression of transposable element transposition. DNA double-strand breaks arising in mutants of the piRNA pathway correlate with affected embryonic axis specification, and this developmental defect is suppressed by mutations in the Chk2 DNA-damage signal transduction pathway. This study found that defects in nos mRNA deadenylation and decay observed in aub or armi mutants were not suppressed by Chk2 (mnkP6) mutations, indicating that these defects did not result from activation of the Chk2 pathway earlier during oogenesis. Moreover, affected deadenylation of nos mRNA in piRNA pathway mutants did not depend on oskar (Rouget, 2010).
A potential direct role of the piRNA pathway in the regulation of nos mRNA deadenylation and decay in the embryo was addressed. Aub and Piwi accumulate in the pole plasm and in pole cells of the embryo. However, lower levels of Aub and Piwi are found throughout the entire embryo. Ago3 is also present throughout the embryo. Aub and Ago3 are found in the cytoplasm and accumulated in discrete foci, a distribution similar to that of CCR4 and Smg. CCR4 and Smg were reported to partially colocalize in small cytoplasmic foci (Zaessinger, 2006). Aub and Ago3 also partially colocalize with Smg and CCR4 in the bulk of syncytial embryos, in both cytoplasmic foci and a diffusely distributed cytoplasmic pool. Importantly, the distributions of CCR4 and Smg depended on the piRNA pathway; they are strongly affected in aub and spn-E mutant embryos. Although global amounts of CCR4 and Smg do not decrease in mutant embryos, CCR4 foci strongly increase in size, whereas Smg foci decreases in size or disappears. This suggests that subsets of CCR4 and Smg foci have different functions and that deadenylation may take place diffusely in the cytoplasm. These results demonstrate a functional link between CCR4-mediated deadenylation and the piRNA pathway (Rouget, 2010).
Co-immunoprecipitation experiments showed that Aub co-precipitate Smg, CCR4 and Ago3 in the absence of RNA, indicating the presence of these proteins in a common complex. Smg also co-precipitates CCR4, Aub and Ago3; however, Piwi is not found to co-precipitate Smg or CCR4. Importantly, Smg, CCR4 and Ago3 also co-precipitate with Aub in osk54 mutant embryos that are defective in pole plasm assembly, indicating the presence of this complex outside the pole plasm. Next it was shown that nos mRNA co-precipitate with Aub in both wild-type and osk54 embryos. The amount of nos mRNA is similar in Aub and Smg immunoprecipitates (Rouget, 2010).
These findings show that the Argonaute proteins Aub and Ago3 associate with Smg and the CCR4 deadenylase complex to directly regulate nos mRNA in the bulk cytoplasm of early embryos (Rouget, 2010).
The nos 3'untranslated region (UTR) contains Smg-binding sites located in its 5'-most region [referred to as the translational control element (TCE)]. piRNAs sequenced from early embryos and presumed capable of targeting nos 3'UTR were sought based on their sequence complementarity. Notably, a specific region located in the 3'-most part of the 3'UTR could be targeted by over 200 copies of piRNAs originating from two transposable elements, 412 and roo. piRNAs complementary to nos 3'UTR were visualized by northern blots. In addition, piRNAs predicted to target nos 3'UTR co-immunoprecipitated with Aub. nos genomic transgenes deleted for different parts of the 3'UTR were used to address the requirement of the corresponding regions for nos mRNA deadenylation. It was shown previously that the TCE (nucleotides 1-184) is required for nos mRNA poly(A) tail shortening, consistent with the role of Smg in this process. Deletion of region 184-403 (nos(delta1)) had no effect, whereas poly(A) tails from the transgene deleted for the region 403-618 (nos(delta2)) were elongated in 3-4-h embryos. This could indicate regulation by the miRNA predicted by miRBase to target this region. Deletion of 618-844 in the nos 3'UTR (nos(delta3)) had a strong effect on nos deadenylation. Consistent with this, nos mRNA levels produced by this transgene remained mostly stable. This resulted in defects in embryo patterning: a total of 35% of embryos from nos(delta3) females did not hatch and among them 86% showed head skeleton defects. Next specific sequences complementary to 412 (15 nucleotides) and roo (11 nucleotides) retrotransposon piRNAs were deleted. These short deletions, either independently or in combination, affected nos mRNA deadenylation (Rouget, 2010).
To support further the role of retrotransposon piRNAs in nos mRNA regulation, 412 and roo piRNAs were blocked by injecting specific 2'-O-methyl anti-piRNA in embryos, and cuticles were recorded as a functional assay of Nos ectopic synthesis at the anterior pole. Injection of anti-piRNA(412) or anti-piRNA(roo) resulted in specific head development defects (Rouget, 2010).
Together, these results provide strong evidence that an interaction between piRNAs and nos mRNA is required for nos mRNA deadenylation and translational repression in the first hours of embryogenesis (Rouget, 2010).
This study has identified a new function of the piRNA pathway in the regulation of maternal mRNAs. Recently, piRNAs derived from the 3'UTRs of cellular transcripts have been identified in gonadal somatic cells, although their biological role has not been clarified. It is proposed that piRNAs, in complex with Piwi-type Argonaute proteins Aub and Ago3, target nos maternal mRNAs and recruit or stabilize the CCR4-NOT deadenylation complex together with Smg. These interactions induce rapid mRNA deadenylation and decay. Thus, activation of mRNA deadenylation represents a new direct mechanism of action for the piRNA pathway with an essential developmental function during the first steps of embryogenesis (Rouget, 2010).
Smg is a general factor for mRNA decay during early embryogenesis. Because Aub and Ago3 are present in a complex with Smg in early embryos, a proportion of Smg mRNA targets could be regulated by the piRNA pathway. Consistent with this, other maternal mRNAs that are destabilized during early embryogenesis are targeted by abundant piRNAs and their deadenylation depends on the piRNA pathway (Rouget, 2010).
These piRNAs involved in gene regulation are generated from transposable element sequences. Although transposable elements have been described to be essential for genome dynamics and evolution, their immediate function within an organism has remained rather elusive. This study provides evidence for a co-evolution between transposable elements and the host genome and reveals the direct developmental function of transposable elements in embryonic patterning, through the regulation of gene expression (Rouget, 2010).
Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far (Jain, 2008). Through a sensitized genetic screen, the Argonaute family member Aubergine (Aub) has now been identified as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. These results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex (Becalska, 2011).
Localization of nos mRNA to the posterior of the Drosophila embryo is critical for patterning of the A-P body axis. Although a cis-acting nos mRNA localization signal has been identified, the complement of trans-acting factors required for assembly of a nos RNP complex competent for posterior localization has remained elusive. From a sensitized genetic screen, Aub was identified as a novel nos mRNA localization factor, Aub interacts with nos mRNA in vivo. Importantly, nos localization was shown to be affected by aub acting downstream of osk mRNA localization, implying independent roles for aub in regulating these two transcripts. Although the role of aub in osk localization appears to be indirect, through a requirement in oocyte microtubule organization, these results suggest that aub plays a more direct role in regulating nos mRNA (Becalska, 2011).
A decrease in aub activity leads to defects in nos mRNA localization and, consequently, in patterning of the A-P axis when nos localization signal redundancy is reduced by removing two localization elements. A similar behavior is observed in rump mutants, which exhibit only weak segmentation defects unless redundantly acting elements are removed from the nos localization signal (Jain, 2008). Presumably, elimination of individual localization signal elements compromises localization by stripping away the contributions of nos localization factors with overlapping functions in nos RNP assembly. Conversely, elimination of multiple nos localization factors should lead to a more severe defect than elimination of an individual factor. Consistent with this prediction, decreasing aub gene dosage in rump mutants also leads to more severe loss of abdominal segments (Becalska, 2011).
In addition to allowing a requirement for aub to be uncovered, the sensitized nos+1+3 background has facilitated the separation of an indirect requirement for aub in osk localization from a more direct requirement in nos localization. Defects in osk regulation and abdominal segmentation are observed only when females are homozygous mutant for aub and not when they are heterozygous. By contrast, defects in nos+1+3 localization are observed when females are heterozygotes for aub mutations. These results are further supported by previous data showing that the ability of ectopically expressed Osk to recruit nos mRNA is compromised in aub−/+ embryos (Becalska, 2011).
Aub has been implicated in the rasiRNA pathway that silences retrotransposons in the germline. However, mutation of squash, which encodes a rasiRNA pathway component that interacts with Aub, has no effect on the nos+1+3 transgene. Interestingly, another rasiRNA pathway component, piwi, has the opposite effect of aub on the nos+1+3 transgene, as heterozygosity for a piwi mutation results in increased segmentation in the sensitized background. Mutations that inactivate the rasiRNA pathway, including aub mutations, activate the DNA damage checkpoint, presumably due to unsuppressed transposon activity. Checkpoint activation disrupts microtubule organization and grk translation, resulting in a failure of axis specification that is thought to lead to subsequent defects in osk mRNA localization. However, the effect of aub mutation on nos+1+3 mRNA localization is independent of the DNA damage pathway, providing further evidence that Aub regulates nos independently of osk. Moreover, these results indicate that Aub function in nos localization is distinct from its function in RNA silencing (Becalska, 2011).
Biochemical experiments indicate nos mRNA forms a complex with Aub in vivo, although whether Aub interacts directly with nos mRNA, or is recruited to the complex by other proteins that bind directly to nos, is not yet clear. Soluble recombinant Aub necessary to distinguish between these possibilities has not been obtained. However, the RNA-dependent co-purification of Aub and Rump, combined with evidence for genetic interactions between aub and rump further supports the contribution of Aub to the formation and/or function of a nos localization RNP complex. Whereas Rump is not concentrated at the posterior of the oocyte, Aub-GFP is localized to the posterior during midoogenesis and continues to accumulate at the posterior pole throughout the later stages of oogenesis when nos becomes localized. Thus, the contributions of Rump and Aub to nos RNP complexes may be dynamic, with both proteins accompanying nos as it is dispersed throughout the oocyte during ooplasmic streaming, but only Aub remaining associated with the nos RNP upon its entrapment at the posterior. Isolation and characterization of the full complement of nos localization factors will be essential to dissect the assembly pathway for nos localization complexes. The isolation of aub in a sensitized genetic screen validates the use of such an approach, in addition to biochemical purification strategies that proved successful for isolation of Rump, for achieving this goal (Becalska, 2011).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.