Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 5

filamentous-actin (F-actin)-coated GRK spheres at the anterior-dorsal region of D. melanogaster oocytes (Kugler et al., 2009). 100% (n = 29) 0% (n = 36)  100% (n = 26) 0% (n = 29)  95.5% (n = 22) 8.3% (n = 12)  94.7% (n = 19) 31.4% (n = 35)  15.6% (n = 32) 78.1% (n = 32)  Figure 3.1.15 KRIMP domains display distinct functions. Using nosgal4VP16 to drive germline-specific expression, the expression level of fusion proteins appears to be variable at different stages of oogenesis. The minimal expression of KRIMP-NT appears 77 sufficient to rescue the perinuclear localisation of AGO3 and MAEL, the expression level and anterior-dorsal localisation of GRK, and karyosome compaction of the oocyte; but not precocious osk mRNA translation in krimp mutant ovary. On the other hand, KRIMPCT only restores the timely expression of OSK protein (green) in krimp mutant egg chamber that overexpresses KRIMP-CT (red), but the remaining mutant phenotypes are not rescued. Although GRK localises to the anterior-dorsal region of the oocyte in krimp mutant ovary habouring KRIMP-CT, GRK protein appears to be deposited as spheroid aggregates. The number of rescued ovarioles for each phenotype is expressed as a percentage of the total number of ovarioles counted. Bar is 10 µm. n, sample size. Since KRIMP-NT appeared sufficient to localise AGO3 to the perinuclear region of germline cells, co-IP was performed to examine for the interaction between both proteins in vitro. Surprisingly, when AGO3-HA was pulled down, KRIMP-NT-MYC was undetectable by immunoblotting (Figure 3.1.16a). A similar experiment was performed with KRIMP-CT and no observable interaction was detected (Figure 3.1.16b). a 78 b Figure 3.1.16 KRIMP-NT and CT not interact with AGO3 in vitro. Fusion proteins are synthesized using TNT® Coupled Rabbit Reticulocyte Lysate System (Promega). MYC-tagged (a) KRIMP-NT and (b) KRIMP-CT not coimmunoprecipitate in the presence of HA-tagged AGO3. The in vitro co-IP data therefore suggest that the presence of the CCCH-type zinc finger motif and coiled-coil domain on KRIMP-NT or TUD domain on KRIMP-CT are not sufficient to promote KRIMP direct interaction with AGO3 in vitro. Although MYC and HA have a protein size of only kilodalton (kDa), it remains possible that fusion proteins have altered physical properties and/or interaction modules. Alternatively, certain bridging factors may be expressed sufficiently in the presence of KRIMP-NT to promote AGO3 perinuclear localisation in the ovary but not in vitro. 79 As described earlier, retroelements were de-repressed in krimp mutant (Figure 3.1.13). To determine which of the motif and/or domains contribute to retroelement repression, semiquantitative RT-PCR was performed on total RNAs prepared from krimp mutant ovary expressing either the NT or CT transgene. The expression of KRIMP-NT in krimp mutant rescued the expression of HeT-A, TAHRE, I-element, and mst40, while the expression of KRIMP-CT rescued the expression of all examined retroelements, except HeT-A (Figure 3.1.17). Figure 3.1.17 KRIMP-NT restores retroelement repression. Semi-quantitative RTPCR of retroelements HeT-A, TAHRE, I-element, and mst40 indicates that the expression of KRIMP-NT in krimp mutant ovary rescues retroelement de-repression. The expression of KRIMP-CT in krimp mutant ovary restores all retroelement repression, except HeT-A. Taking all the observations together, the tudor domain is sufficient to ensure the timely expression of OSK protein, while the coiled-coil domain and/or CCCH-type zinc finger 80 mediates KRIMP genetic interaction with AGO3 and MAEL, as well as regulates oocyte polarity. All of the modules on KRIMP appear to participate in retroelement repression, either singly or in combination, to different extents. 3.2 Nuage mediates piRNA-dependent retroelement silencing 3.2.1 Nuage components mediate retroelement silencing Some nuage components such as SPN-E, AUB, and ARMI have been reported to participate in RNAi to regulate polarity during ovary development and/or retroelement silencing in D. melanogaster (Cook et al., 2004; Vagin et al., 2006). Since retrolements were de-repressed in krimp mutant and KRIMP appeared to interact genetically and physically with other nuage components, the expression of different subtypes of retroelements was examined using semi-quantitative RT-PCR in some nuage mutant ovaries. Retrolements that were examined include mst40, LINEs/non-LTRs HeT-A, TART and I-element (Aravin et al., 2003), and the euchromatic LTR retrotransposon roo (Bowen, 2001). HeT-A and I-element were de-repressed in krimp and mael mutants (Figure 3.2.1a), similar to what has been reported previously in vas, aub, and armi mutants (Savitsky et al., 2006; Vagin et al., 2004; Vagin et al., 2006). TART was de-repressed only in mael and aub mutants, whereas mst40 de-repression was only observed in krimp and mael mutants (Figure 3.2.1a). This difference may have resulted from the dissimilarities between the oogenesis progression defects among the different nuage component mutants. Alternatively, this may indicate differences in element specificity among the 81 nuage components. As for roo, no significant de-repression was observed for all examined nuage component mutants (Figure 3.2.1a and c). The semi-quantitative RTPCR results were confirmed using quantitative RT-PCR (Figure 3.2.1b-c). a b c d Figure 3.2.1 LINEs are de-repressed in the nuage component mutants. (a) Semiquantitative RT-PCR of the retroelements HeT-A, I-element, TART, mst40, and roo in the nuage component mutants. (b-c) Quantitative RT-PCR of the retroelements in the nuage component mutants armi, aub, krimp, and mael. cDNAs that are previously employed for the semi-quantitative RT-PCR in (a) are used for quantitative PCR, in the presence of iQ™ SYBR® Green Supermix (Bio-Rad). A mock reaction without reverse transcriptase is also performed for each RNA sample. All results are normalised with respect to adh. (b) HeT-A is significantly de-repressed in the nuage component mutants krimp and mael, and RNA silencing component mutants aub and armi. (c) I-element is significantly de82 repressed in the nuage component mutants krimp and mael, and RNA silencing component mutants aub and armi. TART is significantly de-repressed in mael mutant. Significant de-repression of mst40 is observed in krimp and mael mutants. No roo derepression is observed in the nuage component mutants. Error bars indicate the standard deviation between the triplicates of each sample. * p < 0.01, ** p < 0.05, n = 3. (d) In tud1 mutants, all the examined retroelements are repressed to similar extents as in the control. RT-PCR was performed using Superscript III One-step RT-PCR kit (Invitrogen). In one of the nuage component mutants tud, HeT-A, I-element, and TART appeared to be repressed to similar extents as the control ovary (Figure 3.2.1d). This suggests that TUD performs a different role at the nuage site. Indeed, a recent study has suggested that TUD aids in the association of piRNAs with AUB and AGO3 in an arginine methylationdependent manner (Nishida et al., 2009). The nuage components, at least for those that were examined, appeared to exhibit different regulatory specificities for different retroelement subtypes. The LINE family elements HeT-A, I-element, and TART, as well as mst40, are located predominantly in the heterochromatin regions of the chromosome, whereas roo is primarily a euchromatic retrotransposon (Aravin et al., 2003; Bowen, 2001). Defective silencing of LINEs/nonLTRs among the examined nuage components therefore implies a common role in maintaining the silenced state of the heterochromatic retroelements. Vagin et al (2004 and 2006) have shown that unlike the nuage components SPN-E, VAS, and AUB, the conventional dicing enzymes (Dicer-1) DCR-1 and DCR-2 are not involved in the silencing of retroelements in the Drosophila female germline. Indeed, immunostaining for the nuage proteins in dcr-1 and dcr-2 mutants indicated normal 83 perinuclear localisation of VAS, AUB, KRIMP, and MAEL (Figure 3.2.2). This suggests that the conventional dicing enzymes and nuage components function independently, and distinct RNA silencing machineries are probably utilised to regulate retroelement MAEL Protein localisation of KRIMP AUB VAS expression in the germline. Figure 3.2.2 Nuage localisation is unaffected in the conventional dicing enzyme mutants dcr-1 and dcr-2. Immunotstaining of VAS, AUB, KRIMP, and MAEL in dcr-1 mutant clones and dcr-2 homozygous mutants indicates normal perinuclear localisation. Bar is 10 µm. 3.2.2 Nuage components regulate the production of piRNAs A previous report has linked the upregulation of retroelements in aub and armi mutants to the failure in piRNA production (Vagin et al., 2006). To determine if the production of piRNAs is compromised in the nuage component mutants, the levels of roo, I-element, 84 and HeT-A piRNAs were analysed. Interestingly, all of the examined piRNAs were reduced in spn-E, vas, krimp, and mael mutants (Figure 3.2.3). This indicates that the nuage regulates the production of piRNAs. Although roo, I-element, and HeT-A piRNAs were reduced in the nuage component mutants, only I-element and HeT-A, which belong to the LINE family, were significantly de-repressed (Figure 3.2.1). This implies that the amount of roo piRNAs that is produced in the nuage component mutants is sufficient to ensure repression. Alternatively, the expression of roo is regulated by mechanism(s) other than piRNA-mediated silencing. However, it remains possible that the reduction of other LTR-derived piRNAs in the nuage component mutants may affect the repression of their respective transcripts. Figure 3.2.3 Production of piRNAs is defective in the nuage component mutants. PAGE northern analysis of roo, I-element and HeT-A piRNAs. The production of roo, Ielement and HeT-A piRNAs is reduced in the nuage component mutants. 85 . at the nuage site. Indeed, a recent study has suggested that TUD aids in the association of piRNAs with AUB and AGO3 in an arginine methylation- dependent manner (Nishida et al., 2009). The. the Drosophila female germline. Indeed, immunostaining for the nuage proteins in dcr-1 and dcr-2 mutants indicated normal 84 perinuclear localisation of VAS, AUB, KRIMP, and MAEL (Figure. silencing of LINEs/non- LTRs among the examined nuage components therefore implies a common role in maintaining the silenced state of the heterochromatic retroelements. Vagin et al (2004 and