Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 10

Figure 3.4.2 Cytoplasmic KRIMP not overlap with PDI-GFP. KRIMP (red) cytoplasmic foci not overlap with PDI-GFP (green), which is a disulfide isomerase that mediates protein folding in the lumen of ER. Bar is 10 μm. The overlaps of pi-bodies with the vesicular bodies suggest that piRNA-mediated retroelement silencing and the endosomal pathway are connected. Hence, it will be interesting to understand in the future how the formation of such cytoplasmic compartments regulates retroelement silencing. 106 Discussion The nuage is a unique, electron-dense structure that is found on the perinuclear regions of many animal germline cells. The evolutionarily conserved nature of the nuage emphasizes its importance and essentiality in the germline. Although the structure of the nuage has been extensively studied since the 18th century, its composition and contribution(s) to the germline are still not well-apprehended. In this thesis, I have described a function for the nuage in mediating post-transcriptional retroelement silencing. The repression of retroelements in the germ cells, which are the founder cells of future generations, is imperative since rampant transposition can inflict deleterious mutations on the genome and compromise gene functions such as those that regulate the host fitness and fertility. An interesting host-derived retroelement function in the Drosophila germline is telomere length maintenance. The telomere length is preserved in the Drosophila germline by the adequate repression of the expression and transposition of telomeric retroelements such as HeT-A, TART, and TAHRE (Savitsky et al., 2006; Vagin et al., 2004). In this course of study, I have demonstrated that the nuage components SPN-E, VAS, AUB, KRIMP, MAEL, and ARMI play a significant role in repressing some telomeric retroelements, as well as other non-LTR and non-telomeric counterparts such as mst40 and I-element. Repression of the retrolements to a physiological level appears to be mediated by a unique class of small RNAs, known as piRNAs. piRNAs are reported to interact with the AGO proteins such as AUB, AGO3 and Piwi, which harbour endoribonucleolytic/slicer 107 functions to promote mRNA cleavage (Brennecke et al., 2007; Gunawardane et al., 2007; Saito et al., 2007). These findings therefore suggest that the nuage silences retroelement expression post-transcriptionally. 4.1 Nuage role in post-transcriptional regulation Using the Drosophila ovary as an in vivo system, I have demonstrated the localisation of the nuage proteins AUB, KRIMP and AGO3, and mRNA degradation enzymes dDCP1/2, Me31B, and PCM in the pi-bodies. The integrity of the pi-bodies appears to be piRNAdependent and correlates with retroelement silencing. This involves contributions from both the nuage and mRNA degradation proteins. By inducing the transcription of an exogenous HeT-A and then examining for decay/stabilisation of the transcript, I further conclude that piRNA-mediated retroelement silencing is in part post-transcriptional in vivo. Moreover, mRNA degradation components DCP1 and SKI3 repress the expression of retroelement HeT-A, without exhibiting noticeable piRNA biogenesis defect. This implies a defect in processes downstream of piRNA production, possibly the removal of the retroelement transcripts by mRNA degradation. In view of past and recent findings, my work suggests that the mRNA degradation machinery mediates the post-transcriptional removal of the retroelement transcripts or decay intermediates, possibly upon piRNA-mediated cleavage (Brennecke et al., 2007; Findley et al., 2003; Harris and Macdonald, 2001; Kennerdell et al., 2002; Li et al., 2009; Lim et al., 2009; Malone et al., 2009). The 5’ and 3’ moieties of the decay intermediates generated by RISC-mediated endoribonucleolytic cleavage are removed by the XRN1 108 and SKI/exosome complexes respectively in S2 cells (Orban and Izaurralde, 2005). However, retroelement decay intermediates are not detected in vivo with the mRNA degradation mutants pcm and ski3 at steady-state. This may reflect the redundancy of other enzymes in the single mRNA degradation mutants in mediating degradation. Alternatively, mRNA degradation genes may contribute to the post-transcriptional silencing of retroelements via a piRNA-independent pathway. The exogenous HeT-A transcript that was expressed by a single heat-shock induction is efficiently silenced in the control ovary, but remains stabilised in the piRNA pathway mutant aub. This suggests that post-transcriptional retroelement silencing by piRNAs occurs in trans. Indeed, the introduction of antisense I-element transgene into the Drosophila female germline results in the silencing of the sense transcript (Gauthier et al., 2000; Jensen et al., 1999a; Jensen et al., 1999b; Malinsky et al., 2000; Robin et al., 2003). Furthermore, trans-silencing of homologous transposons by telomere-associated piRNAs has been reported in D. melanogaster female germline (Josse et al., 2007). Since the HeT-A transgene is placed under the control of an inducible promoter, possible contributions from natural promoters or UTRs in mediating silencing are also ruled out. However, it remains possible that piRNAs are targeted to the nascent transcript and HeTA is silenced co-transcriptionally. 4.2 Nuage role in transcriptional regulation The examination of steady-state retroelement mRNAs shows more substantial accumulation of full-length HeT-A transcript, when compared to the stabilised exogenous 109 HeT-A in aub mutant. This suggests that the destabilisation of HeT-A in wild-type ovary involves an additional hierarchy of regulation besides post-transcriptional control. Indeed, several evidences have suggested and shown that retroelements are silenced transcriptionally (Costa et al., 2006; Kim et al., 2006; Klenov et al., 2007; Pal-Bhadra et al., 2004). In Drosophila ovary, it has been reported that SPN-E represses germline, but not somatic, expression of HeT-A by regulating the chromatin state of retroelement promoter region in a piRNA-dependent manner (Klenov et al., 2007). Mutations in spn-E and aub also impact the de-localisation of HP1 and HP2 from the chromatins (Pal-Bhadra et al., 2004). Moreover, Drosophila MAEL shuttles between the nucleus and cytoplasm (Findley et al., 2003) and mouse MAEL associates with the chromatin remodeler SNF5/INI1 (Costa et al., 2006). Some Drosophila nuage components such as KRIMP and SPN-E contain tudor domains that are implicated to associate with the methylated peptides of histones H3 and H4 (Kim et al., 2006). Hence, piRNA-RISCs may regulate the chromatin state by influencing the localisation or de-localisation of modifying factors to repress unfavourable gene expression in the germline cells. Taken together, at least two hierarchies of retroelement surveillance appear to function in the fly germline, possibly post-transcriptional regulation in the cytoplasm and transcriptional control in the nucleus. 4.3 pi-bodies are linked to endosomal trafficking The association of the cytoplasmic nuage with the mRNA degradation proteins in the Drosophila ovary hints that a macromolecular RNP complex is implicated in the post110 transcriptional retroelement silencing at the pi-bodies. Indeed, other nuage components besides AUB, AGO3, and KRIMP, also localise to the same cytoplasmic nuage bodies (unpublished). Intriguingly, the pi-body function appears to be coupled to the secretory and/or endosomal pathways as observed from the abnormalities between the association of TER94 and endosomal markers with nuage/P-body foci in the piRNA pathway mutants. Moreover, recent interesting works have implicated the interdependency of RNAi and endosomal trafficking (Gibbings et al., 2009; Lee et al., 2009), and TER94 is also found to associate with the nuage component VAS and P-body protein Me31B (Thomson et al., 2008). One of the endosomal markers ARF6 is a monomeric GTPbinding protein that promotes the internalisation of G-protein coupled receptors (Houndolo et al., 2005). Hence, I speculate that specific signaling cascade(s) is activated to target piRNA-RISCs and/or P-bodies upon receptor internalisation. To put endosomal trafficking into the perspective of pi-bodies, this phenomenon may reflect a form of host defense against retroelement infection by localising RNAi machinery to these cytoplasmic sites containing endosomal compartments since retroelement-derived counterparts, RNA viruses, are known to deploy the endocytic pathway for entry and spreading (Lee et al., 2009). Besides sharing a similar morphology and architecture with the vesicular bodies, perinuclear and cytoplasmic nuage also resemble other germline features such as the sponge bodies and pole plasm. Sponge bodies consist of elongated elements formed by ER-like cisternae or vesicles, interspersed in an electron-dense amorphous material (Wilsch-Brauninger et al., 1997). Pole plasm represents a specialised, cytoplasmic region 111 that contains the polar granules, which are posterior determinants of the future PGCs or pole cells. Like the nuage, sponge bodies and pole plasm lack surrounding membranes, contain RNAs, and is frequently associated with the ER and mitochondria. Some nuage components such as VAS and AUB are also detected in the pole plasm (Snee and Macdonald, 2004). The nuage, sponge bodies, and pole plasm may therefore represent intracellular compartments for the assembly and transport of cis- and trans-acting elements involved in RNA silencing. 4.4 The nuage is a multi-protein structure Since the nuage components appear to participate in retroelement silencing as a multiprotein structure, the elucidation of individual gene function(s) will provide insights to how these proteins function mutually as a RNP complex. The mechanistic functions of some nuage components have already been reported: AUB and AGO3 possess endoribonucleolytic activities to cleave mRNA in vitro (Gunawardane et al., 2007); MAEL has promoter binding capability to exert regulation at the transcriptional level (Pek et al., 2009); the intron of VAS encodes for a protein, VAS intronic gene (VIG), that constitutes a component of the RISC (Caudy et al., 2002). One molecule of interest in this thesis is KRIMP, a nuage component that is identified in the laboratory. KRIMP protein contains a CCCH-type zinc finger motif, a coiled-coil domain, and a tudor domain. In the current study, I have characterised krimp mutant phenotypes and shown that it shares similar defects as the other nuage component 112 mutants. These defects include oocyte polarity specification, oocyte karyosome compaction, timely osk mRNA translation during oogenesis, and piRNA-dependent retroelement silencing. The motif and domains of KRIMP exhibit distinct functions as observed from the phenotypic rescue of krimp mutant ovary harbouring different truncated KRIMP transgenes. The expression of tudor domain alone is sufficient to ensure the timely expression of OSK protein. On the other hand, the simultaneous expression of coiled-coil domain and CCCH-type zinc finger motif restores the oocyte polarity defect, as well as KRIMP genetic interaction with AGO3 and MAEL. All of the modules on KRIMP appear to participate in retroelement repression, either singly or in combination, to different extents. To further distinguish the contributions of the coiledcoil domain and CCCH-type zinc finger motif, I have already generated another two transgenes, each harbouring either only the coil-coiled domain or zinc finger motif. The CCCH-type zinc finger motif and tudor domain have been extensively studied in multiple organisms. Proteins with CCCH-type zinc finger motif(s) are thought to exhibit RNA-binding properties and are predominantly described in AU-rich element (ARE)mediated mRNA decay. mRNAs habouring AREs are characterised by the presence of AUUUA motifs within the sequence and are targeted by RNA-binding proteins (Murray and Schoenberg, 2007). For instance, the presence of AREs within tumour necrosis factor alpha (TNFα) mRNA renders its susceptibility to deadenylation by a CCCH-zinc finger protein, Tristetraprolin during inflammation in C. elegans (Lai et al., 1999). Interestingly, two copies and one copy of AUUUA motifs are detected in the 5’- and 3’UTRs of the retroelement HeT-A sequence (unpublished). Hence, it is probable that 113 KRIMP functions as a RNA-binding protein to trigger ARE-mediated retroelement decay. Tudor domains of several proteins such as TDRD1, TDRD2, TDRD4, TDRD6, TDRD7, and TDRD9, are reported to bind the mouse Piwi homologues MIWI and MILI in the germline cells (Vagin et al., 2009). The specificity of tudor-MIWI/MILI interaction depends on the presence of dimethylated arginine residues in MIWI/MILI. Besides mouse Piwi, arginine residues are also dimethylated in Drosophila AUB and AGO3 (Kirino et al., 2009). Protein arginine methyltransferase (PRMT5) is found to associate with Piwi, AUB and AGO3, and is necessary to promote arginine dimethylation, as well as retroelement silencing and piRNA production. This suggests that arginine dimethylation of the AGO proteins by PRMT5 is critical to mediate retroelement repression (Kirino et al., 2009; Vagin et al., 2009). Hence, it will be interesting to determine if KRIMP-AUB/AGO3 interaction is dependent on arginine dimethylation. Lastly, a yeast-2-hybrid screen has identified a E3 ubiquitin ligase complex factor, Speckle-type POZ protein SPOP (also known as Roadkill in D. melanogaster), as a potential interactor of KRIMP (Liu et al., 2009), suggesting that ubiquitinylation has regulatory role(s) in retroelement silencing. Indeed, two recent works have shown that ubiquitinylation is essential to aid in miRNA loading onto the RISC (Gibbings et al., 2009; Lee et al., 2009). In addition, SPOP is highly expressed in a number of cancer cell types, which include liver, kidney, prostate, testes, and uterus (Liu et al., 2009), indicating that KRIMP potentially regulates tumourigenesis. 114 4.5 Future perspectives In my thesis work, I have focused on understanding the nuage’s contributions to retroelement silencing in the female germline of D. melanogaster. Other interesting open questions include the functional conservation of the nuage in DNA transposon silencing, as well as the existence of an analogous somatic nuage counterpart. 4.5.1 Nuage potential role in RNAi of DNA elements It is now evident that the nuage, as well as P-body components, contribute to the silencing of retroelements in the germline of D. melanogaster (Brennecke et al., 2007; Chen et al., 2007; Gunawardane et al., 2007; Lim and Kai, 2007; Lim et al., 2009; Pane et al., 2007; Vagin et al., 2004; Vagin et al., 2006). Since DNA transposons also manifest in the germline (Laski et al., 1986; Rio et al., 1986), it is also exciting to speculate the involvement of the nuage in the silencing of DNA elements. 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One isolated mutant allele pcm∆1 shows a deletion from position 4069 to the end of the coding sequence. (b) Analysis of pcm transcripts in pcm∆1. Semi-quantitative one-step RT-PCR indicates that pcm mRNA is truncated at the 3’ end in pcm∆1. The expression of a neighbouring gene nat1 is unaffected in this mutant allele. (c) Western analysis of PCM protein in pcm∆1. PCM protein is undetectable in pcm∆1, suggesting that the truncated 144 mRNA is not translated. (d) Analysis of ski3 transcripts in ski3f03251. The piggyBac insertion f03251 in the third exon of CG8777 (gene encoding ski3) shows partial lethality for trans-heterozygotes with Df(2R)Np5. Semi-quantitative one-step RT-PCR using primer sets (arrows) that amplify regions before, within or after the insertion, indicates that transcription is disrupted in the trans-heterozygotes. (e) Western analysis of SKI3 protein in ski3f03251. SKI3 protein is undetectable in ski3f03251, suggesting that the truncated mRNA is not translated. 145 6.2 Appendix II Schematic diagram depicting the Directional TOPO cloning (Invitrogen) strategy. TOPO cloning strategy is based on the use of Topoisomerase I, which binds and cleaves the sense strand of the vector. The 3’- end of the cleaved strand is gated by the formation of a covelent bond between the tyrosine 274 on Topoisomerase I and 3’- phosphate of the cleaved DNA. Following the incubation with a double-stranded PCR product that harbours a CACC sequence at the 5’- end, the GTGG overhang in the linearised pENTR™/D-TOPO® invades and displaces the lower strand of the DNA duplex. The phosphor-tyrosyl bond between the vector and Topoisomerase I is reversed and the PCR fragment is cloned in. 146 6.3 Appendix III Schematic diagram depicting site-specific recombination using Gateway® Technology (Invitrogen). Gateway® Technology makes use of the site-specific recombination properties of bacteriophage lambda. pENTR™/D-TOPO® harbouring the gene-of-interest (entry clone) is mixed with a destination vector that contains the ccdB gene. The CcdB protein interferes with DNA gyrase (Bernard and Couturier, 1992). Hence, non-resistant E. coli expressing this protein will not be able to survive. In the presence of Clonase enzyme, site-specific recombination is initiated at the att sites between the entry clone and destination vector. The gene-of-interest is then swapped into the destination vector, which can be isolated by ampicillin selection. Since the expression of ccdB gene is lethal, E. coli carrying the destination or donor vector that lacks the geneof-interest will not be able to grow. 147 6.4 Appendix IV VAS KRIMP VAS krimpf06583/Df WT KRIMP krimpf06583 is a loss-of-function allele. KRIMP (green) and VAS (red) localises to perinuclear nuage in D. melanogaster germline cells. In transheterozygotes, krimp/Df(2R)Exel6063, KRIMP expression is undetectable, indicating that krimpf06583 is a loss-of-function allele. Bar is 10 μm. 148 6.5 Appendix V GAL4 UASp YFP Gene X YFP-tagged Protein X Schematic representation of the yeast UAS/GAL4 expression system. The transcription factor GAL4, binds to the UASp promoter and drives the expression of gene X to generate a fusion protein, YFP-tagged Protein X. GAL4 expression can be induced by placing it under tissue-specific promoters such as nosgal4VP16 (germline-specific in D. melanogaster). 149 6.6 Appendix VI Schematic diagram depicting the ms2/MCP-GFP labeling system. The ms2/MCPGFP labeling system involves the use of two heat-shock-inducible transgenes. One transgene contains the retroelement coding sequence, devoid of the 5’-UTR and promoter region, and fused to six tandem stem-loop binding sites for bacteriophage MCP at the 3’UTR. The other encodes for the fusion protein, MCP-GFP. Both transgenes are introduced into the embryos to generate transgenic flies. F1 progenies arising from the cross between both transgenic fly lines are subjected to heat-shock and MCP-GFP binds the recognition motif on retroelement-(ms2)6 transcripts. 150 6.7 Appendix VII Antisense HeT-A piRNAs localise to GFP-labeled HeT-A bodies. FISH indicates that the signals corresponding to antisense HeT-A piRNAs (red) co-localise with GFP-labeled HeT-A mRNAs (green) in the same cytoplasmic bodies (green arrows) in the control ovary. In krimp mutant ovary where piRNA production is compromised, significant signals corresponding to antisense HeT-A piRNAs are not detected. Bar is 10 μm. 151 [...]... Vagin, F Bantignies, G Cavalli, and V .A Gvozdev 2004 Dissection of a natural RNA silencing process in the Drosophila melanogaster germline Mol Cell Biol a2 4:6742-6750 Aravin, A. 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