Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 7

cytoplasmic bodies is observed. Bars are 20 µm and 10 μm for egg chambers and nurse cells, respectively. 3.3.3 piRNAs are localised to the GFP-labeled HeT-A bodies AUB and AGO3 complexes are reported to associate with piRNAs in the germline (Gunawardane et al., 2007; Nishida et al., 2007). In addition, recombinant AUB and AGO3 are competent for cleaving a substrate RNA at a siRNA target site (Gunawardane et al., 2007), indicating that these AGO proteins are potential constituents of germline RISCs. In order to determine whether the nuage cytoplasmic foci consist of piRNAloaded germline RISCs, the presence and localisation of antisense piRNAs were examined using FISH. The antisense HeT-A piRNA signals co-localised with the GFPHeT-A foci in the aub control ovary (green arrows, Figure 3.3.5a and a’), while an irrelevant probe hybridising to antisense 2S rRNA or a piRNA-unrelated HeT-A antisense region did not exhibit any significant co-localisation (Figure 3.3.5b and b’, and data not shown). A control staining with secondary antibodies alone did not exhibit non-specific labeling of the GFP-HeT-A foci (data not shown), further indicating the specificity of HeT-A piRNA probe. In aub mutant where the production of piRNAs is impaired (Vagin et al., 2006), the antisense HeT-A piRNA signal was undetectable (Figure 3.3.5c). Similarly, specific antisense HeT-A piRNA signal was observed in the control but not in krimp mutant ovary (Appendix VII). The localisation of both piRNAs and the target transcripts to common foci that possibly contain the mRNA degradation proteins reflect the assembly of a macro-RNP complex committed to the removal of retroelement transcripts. To distinguish them from conventional P-bodies, these foci are termed as pibodies. 92 a a’ b b’ c Figure 3.3.5 Antisense piRNAs co-localise with GFP-HeT-A bodies. (a) FISH for antisense HeT-A piRNAs in aub mutant. In the aub control ovary, antisense HeT-A piRNAs co-localise with GFP-labeled HeT-A mRNA in the same cytoplasmic bodies (green arrows in a’). (b and b’) FISH with the control probe against antisense 2S rRNA does not co-localise with GFP-labeled HeT-A mRNA. (c) In aub mutant, antisense HeT-A piRNA signal is undetectable. Bar is 10μm. 3.3.4 pi-body assembly is piRNA-dependent and correlates with retroelement silencing In spn-E and aub mutants where the production of piRNAs is compromised (Vagin et al., 2006), no overlaps between cytoplasmic KRIMP foci and dDCP1/2, Me31B or PCM were observed (Figure 3.3.4a and Figure 3.3.6), indicating that pi-body assembly is disrupted. This observation suggests that cytoplasmic nuage proteins and the targeted mRNAs are recruited to mRNA degradation components, or vice versa, in a piRNAdependent manner, forming pi-bodies that can contribute to retroelement silencing. 93 Figure 3.3.6 Assembly of the pi-bodies is piRNA-dependent. In the wild-type ovary, mRNA degradation proteins dDCP1/2, Me31B, and PCM, overlap cytoplasmic KRIMP foci. In contrast, in piRNA pathway mutants spn-E and aub, where the production of piRNAs is compromised, cytoplasmic KRIMP foci no longer overlap with the mRNA degradation proteins. Bar is 10 µm. To exclude the possibility that the failure of pi-body assembly in spn-E and aub mutants impairs the activities of mRNA degradation proteins and results in retroelement silencing, two representative activities of mRNA turnover, namely deadenylation and decapping of a non-retroelement transcript cycB, were examined. The rapid degradation of the cyc 94 transcripts has been shown to be regulated by deadenylation (Morris et al., 2004). To monitor deadenylation activity, the length of the cycB poly(A) tail was examined in the aub mutant using LM-PAT assay (Figure 3.3.7; Salles et al., 1999). In comparison to the deadenylase mutant twin, where deadenylation was compromised and longer poly(A) tails were observed, cycB transcript appeared to be efficiently deadenylated in the aub mutant. This indicates that deadenylation is unaffected in at least one of the piRNA pathway mutant aub. Figure 3.3.7 Deadenylation is unaffected in aub mutant. LM-PAT assay of cycB. In the piRNA pathway mutant aub, deadenylation appears unaffected since the poly(A) tail length is comparable to that of the control. In contrast, deadenylation is impaired in the deadenylase mutant twin, as indicated by the accumulation of longer poly(A) tails. To examine decapping, total RNAs extracted from both control and aub mutant ovaries were treated with Terminator™ 5’-phosphate-dependent exonuclease, and subjected to 95 RT-PCR for cycB. RNA molecules with 5’ cap structures are protected from the exonuclease and therefore competent for amplification. Similar to the control, cycB transcript was comparably degraded by the exonuclease in the aub mutant (Figure 3.3.8), indicating that cycB mRNA is efficiently decapped. Figure 3.3.8 Decapping is unaffected in aub mutant. Cap analysis of cycB. In aub mutant as well as in the control, the 5’-UTR of cycB is efficiently degraded by Terminator™ 5’-phosphate-dependent exonuclease, indicating that decapping of cycB takes place normally. The control small-nuclear RNA U1, which harbours a 2,2,7trimethyl G-cap, is resistant to exonuclease treatment. Consistent with the unaffected mRNA degradation activities in aub mutant, no observable changes in the protein expression of four mRNA degradation proteins PCM, SKI3, Me31B, and dDCP1 were detected in aub or krimp mutants (Figure 3.3.9). Taken together, these data show that the retroelement silencing defect in the piRNA pathway mutants is not due to impairment of the mRNA degradation machinery. 96 Figure 3.3.9 The protein expression of mRNA degradation proteins is unaffected in piRNA pathway mutants. Western analyses of mRNA degradation protein expression in the piRNA pathway mutants. Expression of DCP1, SKI3, PCM, and Me31B is unaffected in aub and krimp mutants. 3.3.5 piRNA-mediated retroelement silencing is post-transcriptional The localisation of the retroelement transcripts with the nuage/P-body foci suggests that retroelement silencing is post-transcriptional. To determine if piRNA-mediated retroelement silencing is post-transcriptional in vivo, retroelement decay was investigated in aub control and mutant ovaries using the inducible HeT-A transgene harbouring ms2. Following the induction of HeT-A ms2 mRNA by heat-shock, ovaries were dissected at time intervals up to 12hr, and northern blotting was performed with a probe that specifically detects exogenous HeT-A ms2. In the control ovary, HeT-A ms2 mRNA was 97 rapidly degraded and the level of transcript was undetectable by 100 post-induction (Figure 3.3.10). On the other hand, in the aub mutant, HeT-A mRNA exhibited prolonged stabilisation (Figure 3.3.10) and remained detectable to days post-induction (data not shown). This suggests that a prolonged post-transcriptional retroelement silencing is dependent on piRNAs. a b Figure 3.3.10 Post-transcriptional retroelement silencing is piRNA-dependent. (a) Timecourse of HeT-A ms2 mRNA decay in the aub mutant. HeT-A ms2 expression is induced by heat-shock and changes in mRNA abundance are measured over time. A rapid initial degradation of HeT-A ms2 mRNA is observed in both control and aub mutant ovaries. However, aub mutant exhibits prolonged stabilisation of the HeT-A MS2 transcript, while the exogenous mRNA is undetectable in control ovaries by 100 mpi. (b) Graphical output of normalised HeT-A ms2 transcript against gadph mRNA in control and aub mutant ovaries. Error bars indicate the standard deviation between the triplicates of each sample. n = 3. The examination of endogenous retroelement transcripts also showed significant accumulations of full-length HeT-A and I-element mRNAs in spn-E, aub, and krimp mutants (Figure 3.3.11). In addition, a low level of full-length HeT-A transcript 98 . reported to associate with piRNAs in the germline (Gunawardane et al., 20 07; Nishida et al., 20 07) . In addition, recombinant AUB and AGO3 are competent for cleaving a substrate RNA at a siRNA target. tail length is comparable to that of the control. In contrast, deadenylation is impaired in the deadenylase mutant twin, as indicated by the accumulation of longer poly (A) tails. To examine. cycB poly (A) tail was examined in the aub mutant using LM-PAT assay (Figure 3.3 .7; Salles et al., 1999). In comparison to the deadenylase mutant twin, where deadenylation was compromised and longer