accumulated in the deadenylase mutant twin, indicating the involvement of at least one of the mRNA degradation enzymes in the post-transcriptional silencing of retroelements, either in a piRNA-dependent or -independent manner. Figure 3.3.11 Full-length retroelement transcripts accumulate in the piRNA pathway and deadenylase mutants in vivo. Northern blotting of endogenous retroelement transcripts in the piRNA pathway and mRNA degradation mutants. Fulllength HeT-A and I-element transcripts accumulate in spn-E, aub, and krimp mutants at steady-state. A low level of full-length HeT-A mRNA accumulates in the deadenylase mutant twin. No detectable full-length retroelement transcript or decay intermediates are observed in the mRNA degradation mutants dcp1, ski3, and pcm. In dcp1, ski3 or pcm mutants, neither of the full-length endogenous retroelements or decay intermediates were detected (Appendix I and Figure 3.3.11). However, a more sensitive quantitative assay RACE-PAT/RT-PCR detected de-repression of the CDS, UTRs and poly(A) regions of HeT-A mRNA in dcp1 and ski3 mutants (Figure 3.3.12), 99 confirming that removal of the retroelement transcripts and/or the decay intermediates involves mRNA degradation enzymes. a 100 b Figure 3.3.12 HeT-A is de-repressed in dcp1 and ski3 mutants. (a) Semi-quantitative RACE-PAT and RT-PCR analyses of retroelement expression in the mRNA degradation mutants. The schematic diagram shows primer sets (arrows) that are used to amplify regions on HeT-A; 5’-UTR, CDS, 3’-UTR, and poly(A). All examined regions are derepressed in dcp1 and ski3 mutants. (b) Quantitative RACE-PAT and RT-PCR of HeT-A mRNA, normalised against act5C, in dcp1 and ski3 mutants. Error bars indicate the standard deviation between the triplicates of each sample. * p< 0.05, **p< 0.01, n = 3. To determine if the mRNA degradation enzymes contribute to retroelement silencing by regulating piRNA production, northern analyses were performed to look at the level of piRNA production in twin, dcp1, ski3, and pcm control heterozygotes and mutant ovaries. The blotting results showed no obvious reduction of HeT-A and I-element antisense piRNAs in the examined mRNA degradation mutants as compared to their respective control heterozygotes (Figure 3.3.13). 101 Figure 3.3.13 piRNA production is unaffected in the mRNA degradation mutants. PAGE-northern analyses of piRNA production in the mRNA degradation mutants. The level of piRNA production is unaffected in the mRNA degradation mutants twin, dcp1, ski3, and pcm. Furthermore, the piRNA pathway proteins AUB, AGO3, and KRIMP exhibited normal perinuclear and cytoplasmic localisation in the mRNA degradation mutants (Figure 3.3.14). The normal piRNA production and nuage localisation therefore suggest that these enzymes not contribute to and/or regulate piRNA biogenesis. Alternatively, they may act in processes that are downstream of piRNA production. Taken together, germline cells contain cytoplasmic pi-bodies, which consist of the retroelement transcripts, antisense piRNAs, and proteins involved in piRNA biogenesis, as well as mRNA degradation. The assembly of pi-bodies is piRNA-dependent and appears to correlate with retroelement silencing. Retroelement silencing is in part posttranscriptional and requires the contribution from both the piRNA pathway and mRNA degradation proteins. 102 Figure 3.3.14 Nuage localisation is unaffected in the mRNA degradation mutants. Immunostaining of the nuage components in mRNA degradation mutants. AUB, AGO3, and KRIMP localisation to both the perinuclear regions and cytoplasmic bodies appear unaffected in the mRNA degradation mutants twin, dcp1, ski3, and pcm. Bar is 20 µm. 103 . accumulated in the deadenylase mutant twin, indicating the involvement of at least one of the mRNA degradation enzymes in the post-transcriptional silencing of retroelements, either in a. degradation proteins. 103 Figure 3.3.14 Nuage localisation is unaffected in the mRNA degradation mutants. Immunostaining of the nuage components in mRNA degradation mutants. AUB, AGO3, and. production in the mRNA degradation mutants. The level of piRNA production is unaffected in the mRNA degradation mutants twin, dcp1, ski3, and pcm. Furthermore, the piRNA pathway proteins AUB, AGO3,