a b c d Figure 3.1.9 UASp-krimp-venus transgene fully rescues krimp mutant defects. By crossing flies harbouring the venus-krimp transgene into krimp mutant background, (a) the oocyte nucleus compacts into a karyosome (blue) and C(3)G (red) becomes chromosomal. Bar is µm. (b) MAEL and AGO3, whose localisation depends on KRIMP, localises to the perinuclear regions of the germline cells. Bar is 10 µm. (c) osk mRNA is repressed normally. Bar is 20 µm. (d) GRK expression is comparable to the wild-type ovariole and the protein localises to the anterior-dorsal region of the stage egg chamber. Bar is 20 µm. 70 3.1.3 KRIMP interacts genetically with other nuage components Previous work has suggested that the nuage components interact genetically: localisation of AUB to the nuage depends on VAS, and MAEL localisation depends on VAS and AUB (Findley et al., 2003). To determine if KRIMP exhibits genetic interaction with other nuage components, KRIMP localisation to the perinuclear nuage was examined in squ, spn-E, vas, aub, cuff, and mael mutants. In all of the examined mutants except mael, KRIMP perinuclear localisation was affected, while the localisation of AGO3 and MAEL appeared to depend on KRIMP, as well as on SQU, SPN-E, VAS, AUB, and CUFF (Figure 3.1.10; Findley et al., 2003). Moreover, AGO3 and MAEL localisation to the perinuclear nuage were rescued in 100% (n = 30) and % (n = 29) of the ovarioles in the presence of a UASp-krimp-venus transgene that was driven by nosgal4VP16 in krimp mutant (Figure 3.1.9b), thereby confirming the dependency of both proteins on KRIMP expression. It was also noticeable that VAS localisation depends partially on proper AUB localisation. Although VAS foci were apparent in aub mutant, cytoplasmic VAS was visibly more abundant than in the wild-type (Figure 3.1.10). Hence, a feedback mechanism may exist between VAS and AUB. The genetic interaction among the different nuage components suggests that there may exist a “platform” that lies in the perinuclear vicinity to facilitate the recruitment of multiple nuage components. It is possible that these examined components have comparable activities in a common molecular machine; or alternatively, they have distinct functions that are carried out using a common platform, the nuage. 71 Figure 3.1.10 Nuage components interact genetically with one another. Ovaries from different mutant flies are immunostained for the nuage components. Homozygous mutant alleles or allelic combinations are used for all the mutants. Localisation of the nuage components at the perinuclear regions of the germline cells reflects a hierarchical assembly. All the nuage components VAS, AUB, KRIMP, AGO3, and MAEL, depend on SQU to localise normally to the perinuclear regions. AUB, KRIMP, AGO3 and MAEL depend on SPN-E and VAS for proper localisation; KRIMP, AGO3 and MAEL depend on AUB and CUFF to localise to the nuage; AGO3 and MAEL depend on VAS, AUB, and KRIMP to localise normally. Bar is 10 µm. 3.1.4 KRIMP interacts physically with other nuage components The genetic dependency of the nuage components on one another, as well the colocalisation of different proteins, is suggestive of the presence of a macromolecular complex or sub-complexes at the perinuclear regions of the germline cells. To determine if the nuage components are functioning as complexes, the physical interactions between in vitro translated KRIMP and some nuage components including AUB, CUFF, AGO3, and MAEL were examined. When KRIMP-MYC was pulled down, AUB-HA, CUFF72 HA, and AGO3-HA were co-immunoprecipitated, indicating that KRIMP interacts directly with these three nuage components (Figure 3.1.11). On the other hand, MAELHA was not, or weakly, pulled down with KRIMP-MYC, suggesting that the nuage is made up of sub-complexes. * Figure 3.1.11 KRIMP interacts directly with AUB, CUFF, and AGO3. Fusion proteins are synthesized using TNT® Coupled Rabbit Reticulocyte Lysate System (Promega). MYC-tagged KRIMP is immunoprecipitated with IgG-coupled Protein A/G beads, in the presence of either HA-tagged AUB, CUFF, AGO3 or MAEL. KRIMPMYC interacts directly with AUB-HA, CUFF-HA, and AGO3-HA. The asterisk indicates the IgG band. To confirm KRIMP association with the nuage components in vivo, KRIMP and one of the interacting nuage components AGO3 were, respectively, expressed as a fusion protein 73 of Venus and HA in the wild-type ovary using the UAS/GAL4 expression system. Coimmunoprecipitation was then performed with lysates prepared from the ovaries. VenusKRIMP co-immunoprecipitated with AGO3-HA (Figure 3.1.12), implying that KRIMP interacts physically with at least one or more nuage components in the germline. Figure 3.1.12 KRIMP interacts with AGO3 in vivo. Venus-tagged KRIMP and HAtagged AGO3 are overexpressed in wild-type ovary using the UAS/GAL4 system and lysates are prepared for co-immunoprecipitation. KRIMP-Venus co-immunoprecipiates with AGO3-HA in ovary lysates. 3.1.5 KRIMP participates in retroelement repression Past work has shown that some nuage components including SPN-E, AUB, and VAS repress the expression of retroelements in the germline (Savitsky et al., 2006; Vagin et al., 2004; Vagin et al., 2006). To investigate if KRIMP has a similar function, semiquantitative RT-PCR was performed on total RNA isolated from krimp control and mutant ovaries. Retroelements that were examined include the repetitive long interspersed nuclear elements (LINEs) such as HeT-A, TAHRE, and I-element (Aravin et al., 2003), and a tandem-repeat that lies near the β-heterochromatin mst40 (Steven and 74 Russel, 1994). In krimp mutant ovary, all of the examined retroelements were derepressed (Figure 3.1.13), suggesting that KRIMP shares a common function with SPNE, AUB, and VAS. Figure 3.1.13 Retroelements are de-repressed in krimp mutant. Semi-quantitative RTPCR of the retroelements HeT-A, TAHRE, I-element, and mst40 in krimp mutant ovary. All examined retroelements are de-repressed in krimp mutant. 3.1.6 KRIMP’s domains display distinct functions To further characterise KRIMP functions, as well as its genetic and physical interactions with other nuage components during oogenesis, truncated N-terminus (NT) and Cterminus (CT) KRIMP transgene variants were generated. KRIMP-NT harboured both the coiled-coil domain and CCCH-type zinc finger motif, while KRIMP-CT only contained the tudor domain (Figure 3.1.14). These transgenes were introduced into krimp 75 mutant and expressed using UAS/GAL4 expression system. Ovaries were then dissected and examined for krimp mutant phenotypes that were rescued. Figure 3.1.14 Schematic drawing depicting KRIMP transgene variants. KRIMP-NT harbours the coiled-coil domain and CCCH-type zinc finger motif. KRIMP-CT only contains the tudor domain. ZnF, zinc finger. KRIMP-NT, when expressed, localised predominantly as perinuclear foci, while KRIMPCT appeared diffuse in the cytoplasm of wild-type ovary (Figure 3.1.15). In krimp mutant ovary that expressed KRIMP-NT, krimp mutant phenotypes including AGO3 and MAEL perinuclear localisation, GRK expression level and anterior-dorsal localisation, and karyosome formation, were rescued. On the other hand, the introduction of the transgene variant for KRIMP-CT into krimp mutant background only rescued the precocious translation of osk mRNA (Figure 3.1.15). Although the anterior-dorsal localisation of GRK appeared to be partially rescued in krimp mutant ovary expressing KRIMP-CT, ectopic GRK spheroid aggregates were detected. This phenocopies bicaudal-C mutant, which is known to affect the secretory pathway and cause the accumulation of 76 . nuage was examined in squ, spn-E, vas, aub, cuff, and mael mutants. In all of the examined mutants except mael, KRIMP perinuclear localisation was affected, while the localisation of AGO3 and. mutants. Localisation of the nuage components at the perinuclear regions of the germline cells reflects a hierarchical assembly. All the nuage components VAS, AUB, KRIMP, AGO3, and MAEL, depend. beads, in the presence of either HA-tagged AUB, CUFF, AGO3 or MAEL. KRIMP- MYC interacts directly with AUB-HA, CUFF-HA, and AGO3-HA. The asterisk indicates the IgG band. To confirm KRIMP association