FUNCTIONAL ANALYSIS OF THE NUAGE, A UNIQUE GERMLINE ORGANELLE, IN DROSOPHILA MELANOGASTER AI KHIM LIM (B Sci (Hons), UNSW) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my sincere gratitude to: My supervisor, Dr Toshie Kai, for her conscientious guidance and supervision throughout my graduate studies My thesis committee members, Dr Stephen Cohen, Dr Frederic Berger, and Dr Cythia He, for their kind advices, stimulating ideas, and critical reading of my manuscripts Toshie’s present and past laboratory members, Liheng Tao, Junwei Pek, Veena Patil, Amit Anand, Junichi Honda, and Annabelle Chen, for providing assistance and feedbacks on my projects P Lasko (McGill University, Montreal, Quebec, Canada), H Ruohola-Baker (University of Washington, Institute for Stem Cell and Regenerative Medicine, Seattle, Washington), A Spradling (Carnegie Institution of Washington, Baltimore, USA), A Nakamura (RIKEN Center for Developmental Biology, Kobe, Osaka, Japan), P M MacDonald (The University of Texas, Austin, USA), D St Johnson (The Wellcome/CRC Institute, Cambridge, United Kingdom), T B Chou (Institute of Molecular and Cellular Biology, Taipei, Taiwan), S Newbury (Brighton and Sussex Medical School, Brighton, United Kingdom), J Wilhelm (University of California, San Diego, La Jolla, CA), R M Long (Albert Einstein College of Medicine, Bronx, New York), and the Drosophila Stock Center for the fly stocks and antibodies, and especially to H Han (McGill University, Montreal, Quebec, Canada) for her unpublished anti-AUB TLL facilities, Media prep, IT department, and Microscopy department, for their support in the flyfood preparation and image acquisition My spouse, You Keat Chia, for his boundless love and encouragement during my hustling work hours My TLL friends, Woei Chang Liew, Alex Chang, and Kaichen Chang, for sharing my laughter and troubles My climbing and diving friends, for bringing joy to my graduate student life My family members, for tolerating my bad days Table of Contents Summary I List of Tables III  List of Figures IV  List of Abbreviations VII  1  Introduction 1  1.1  Drosophila melanogaster germline as a model system 4  1.2  The nuage 9  1.2.1  Oocyte polarity 14  1.2.2  Gametogenesis 14  1.2.3  Post-transcriptional and transcriptional silencing 15  1.3  Retroelements and Piwi-interacting RNAs 16  1.3.1  Retroelements 17  1.3.2  Piwi-interacting RNAs 17  1.3.3  Retroelements/piRNAs host-derived functions 21  1.3.3.1  1.3.3.2  Female fertility control and hybrid dysgenesis in D melanogaster 23  1.3.3.3  1.4  Telomere maintenance 21  Male fertility control in D melanogaster 24  Post-transcriptional gene silencing and Processing bodies 25  1.4.1  Processing bodies 26  1.4.2  mRNA degradation 26  1.4.3  RNA interference 28  1.5  RNA silencing and endosomal trafficking 29  1.6  2  Thesis overview 32  Materials and Methods 34  2.1  Molecular work 34  2.1.1  Recombinant DNA methods 34  2.1.1.1  Strains and culture conditions 34  2.1.1.2  Bacterial glycerol stocks 34  2.1.1.3  Plasmid DNA preparation 35  2.1.1.4  Polymerase Chain Reaction (PCR) 35  2.1.1.5  Restriction digestion 35  2.1.1.6  Sequencing 35  2.1.2  Bacterial transformation 36  2.1.2.1  Preparation of heat-shock competent cells 36  2.1.2.2  Preparation of electrocompetent cells 36  2.1.2.3  Transformation 37  2.1.3  Cloning strategies and constructs 37  2.1.3.1  Conventional cloning 37  2.1.3.2  Gateway® cloning 38  2.1.3.3  TA cloning 39  2.1.4  Single-fly PCR 39  2.1.4.1  Preparation of fly genomic DNA 39  2.1.4.2  Genomic DNA PCR 40  2.1.5  Total RNA extraction 40  2.1.6  Poly A+ RNA purification 40  2.1.7  DNase treatment 40  2.1.8  Reverse transcription (RT) 40  2.1.9  Semi-quantitative and quantitative PCR 41  2.1.10  Poly(A) tail test (PAT) 41  2.1.10.1  Rapid Amplification of cDNA Ends-PAT (RACE-PAT) 41  2.1.10.2  Ligation-mediated PAT (LM-PAT) 42  2.1.11  Decapping assay 42  2.2  Fly genetics 42  2.2.1  Fly husbandry and stocks 42  2.2.2  Generation and clean-up of mutant alleles 44  2.2.3  Generation of transgenic flies by microinjection 44  2.3  Immunohistochemistry and Microscopy 45  2.3.1  Antibody staining of fixed ovaries 45  2.3.2  piRNA Fluorescence in situ Hybridisation (FISH) 47  2.3.2.1  In vitro transcription of RNA probes 47  2.3.2.2  FISH 47  2.3.3  2.4  Microscopy and image processing 48  Biochemistry 48  2.4.1  Recombinant protein expression and purification 48  2.4.2  Antibody generation and affinity purification 49  2.4.3  Immunological detection of proteins 50  2.4.4  Co-immunoprecipitation (co-IP) of protein complexes 52  2.4.4.1  In vivo co-IP 52  2.4.4.2  2.4.5  2.5  In vitro co-IP 53  Northern analyses of transcripts and piRNAs 53  ms2/MCP-GFP labeling system of mRNAs 55  2.5.1  ms2/MCP-GFP labeling of retroelement transcripts 55  2.5.2  Visualisation of artificial retroelement transcripts 55  2.5.3  Timecourse (pulse-chase) of artificial HeT-A transcript 56  2.6  3  Primers 56  Results 59  3.1  Characterisation of a novel nuage component Krimper (KRIMP) 59  3.1.1  KRIMP is a nuage component 59  3.1.2  krimp mutant exhibits spindle-class phenotype 63  3.1.3  KRIMP interacts genetically with other nuage components 71  3.1.4  KRIMP interacts physically with other nuage components 72  3.1.5  KRIMP participates in retroelement repression 74  3.1.6  KRIMP’s domains display distinct functions 75  3.2  Nuage mediates piRNA-dependent retroelement silencing 81  3.2.1  Nuage components mediate retroelement silencing 81  3.2.2  Nuage components regulate the production of piRNAs 84  3.3  Nuage and P-bodies regulate post-transcriptional retroelement silencing 86  3.3.1  Nuage cytoplasmic bodies overlap with mRNA degradation components 86  3.3.2  Retroelement transcripts are localised to the nuage cytoplasmic bodies 89  3.3.3  piRNAs are localised to the GFP-labeled HeT-A bodies 92  3.3.4  pi-body assembly is piRNA-dependent and correlates with retroelement silencing 93  3.3.5  3.4  4  piRNA-mediated retroelement silencing is post-transcriptional 97  Nuage and endosomal trafficking 104  Discussion 107  4.1  Nuage role in post-transcriptional regulation 108  4.2  Nuage role in transcriptional regulation 109  4.3  pi-bodies are linked to endosomal trafficking 110  4.4  The nuage is a multi-protein structure 112  4.5  Future perspectives 115  4.5.1  Nuage potential role in RNAi of DNA elements 115  4.5.2  Does the nuage function in the soma? 116  5  Bibliography 118  6  Appendices 144  6.1  Appendix I 144  6.2  Appendix II 146  6.3  Appendix III 147  6.4  Appendix IV 148  6.5  Appendix V 149  6.6  Appendix VI 150  6.7  Appendix VII 151  Summary Nuage is an electron-dense perinuclear structure that is known to be a hallmark of the animal germline cells Although the conservation of the nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germline still remain unknown In this thesis, I report a novel nuage component Krimper (KRIMP) in Drosophila melanogaster, and show that it ensures the repression of retroelements in the female germline krimp loss-of function allele exhibits female sterility, defects in karyosome formation and oocyte polarity, and precocious oskar translation These phenotypes are commonly observed in two other nuage component mutants spindle-E and aubergine, which are also known to mediate RNA interference This therefore suggests a shared underlying defect that utilises RNA silencing In the nuage component mutants, retroelements HeT-A, TART, I-element, and mst40 are derepressed to different extents De-repression of retroelements appears to correlate with the down-regulation of a unique class of small RNAs, termed as P-element-induced wimpy testes (Piwi)-interacting RNAs (piRNAs) This therefore suggests that the nuage functions as a specialised “centre” to govern genome fidelity in the germline cells via piRNA-mediated gene regulation Besides localising to the perinuclear sites, the nuage/piRNA pathway components are found in cytoplasmic foci that also contain retroelement transcripts, anti-sense piRNAs, and proteins involved in mRNA degradation These mRNA degradation proteins Decapping Proteins, Maternal Expression at 31B (Me31B, a decapping activator), and I Pacman, are normally thought of as components of processing bodies In spindle-E and aubergine mutants that lack piRNA production, piRNA pathway proteins no longer overlap mRNA degradation proteins Concomitantly, spindle-E and aubergine mutant ovaries show an accumulation of full-length retroelement transcripts and prolonged stabilisation of HeT-A mRNA, supporting the role of piRNAs in mediating posttranscriptional retroelement silencing HeT-A mRNA is de-repressed in mRNA degradation mutants, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates II somatic cells and are associated with Piwi (adapted from Siomi and KuramochiMiyagawa, 2009) Recent work has demonstrated that germline and somatic piRNAs are generated by distinct pathways in the Drosophila ovary (Li et al., 2009; Malone et al., 2009) In the ovarian germline cells, piRNAs are produced by a slicer-, AGO3- and AUB-dependent feed-forward amplification loop (Figure 1.3.1 and Figure 1.3.2; Brennecke et al., 2007; Gunawardane et al., 2007; Vagin et al., 2006) The feed-forward amplification loop generates sense and antisense piRNAs through repetitive cycles of binding and cleavage of the sense and antisense retroelement transcripts by antisense piRNA-associated Piwi/AUB and sense piRNA-associated AGO3, respectively (Figure 1.3.1b) The specificity of retroelement binding is determined by the 5’ends of piRNAs, where a uridine (U) and adenine (A) residues are present at the 10th position from the 5’ ends of antisense and sense piRNAs, respectively (Figure 1.3.2, Brennecke et al., 2007; Gunawardane et al., 2007) Hence, the first ten nucleotides of the AUB/Piwi-interacting piRNAs are complementary to that of AGO3-interacting piRNAs and this determines the position at which the target transcripts are cleaved by AUB/Piwi/AGO3 (Brennecke et al., 2007; Gunawardane et al., 2007) On the other hand, somatic piRNAs such as flamenco are generated in an AGO3- and ping pong cycle-independent manner (Figure 1.3.1; Li et al., 2009; Malone et al., 2009) The functional significance of having two distinct piRNA pathways for the germline and somatic cells in the ovary is still not well understood 19 Figure 1.3.2 Ping pong model for piRNA biogenesis Sense piRNA-associated AGO3 binds and cleaves the antisense transcript at the 10th nucleotide, U The antisense piRNA precursor mRNA then associates with AUB and its 3’ end is trimmed by unknown 20 exoribonucleases Antisense piRNA-associated AUB/Piwi then binds and cleaves the sense transcript at the 10th nucleotide, A The cleaved sense transcript then associates with AGO3 and gets trimmed (adapted from Brennecke et al., 2007) 1.3.3 Retroelements/piRNAs host-derived functions Several nuage proteins including VAS, AUB, AGO3, ZUC, SQU, and MAEL are reported to regulate the production of piRNAs (Brennecke et al., 2007; Chen et al., 2007; Gunawardane et al., 2007; Li et al., 2009; Lim and Kai, 2007; Pane et al., 2007; Soper et al., 2008; Vagin et al., 2004; Vagin et al., 2006) The sufficient production of piRNAs in the Drosophila germline is linked to retroelement silencing since these RNA elements are de-repressed in the nuage component mutants vas, aub, ago3, mael, zuc, and squ when piRNA production is compromised (Brennecke et al., 2007; Chen et al., 2007; Gunawardane et al., 2007; Li et al., 2009; Lim and Kai, 2007; Pane et al., 2007; Soper et al., 2008; Vagin et al., 2004; Vagin et al., 2006) Although the host has evolved a piRNAmediated silencing mechanism to safeguard the genome against retroelement invasion, some retroelements have managed to domesticate and co-evolve with the hosts, in that cellular genes derived from these elements are expressed and tightly regulated to aid in both germline and somatic functions 1.3.3.1 Telomere maintenance Unlike eukaryotes, D melanogaster does not rely on telomerase to maintain telomere length Instead, the chromosome ends of D melanogaster are made up of arrays of nonLTR retroelements HeT-A, TART, and TAHRE in both the germline and somatic cells (reviewed in Casacuberta and Pardue, 2006; Frydrychova et al., 2009) In somatic but not 21 germline cells, a LTR-type retroelement Invader4 makes up the subtelomeric ends (Figure 1.3.3; Phalke et al., 2009) Figure 1.3.3 Schematic representation of chromosome 3R telomere-associated sequences in D melanogaster Non-LTR retroelements HeT-A and TART make up the telomeric regions In the subtelomeric regions, six arrays of 943 bp repeats (light grey boxes) are found proximal to HeT-A and TART Each 943 bp repeat consists of a 381 bp telomere-specific repeat (white box) and three copies of defective Invader4 LTR (adapted from Phalke et al., 2009) It has been demonstrated that proteins that are specialised for RNAi such as SPN-E and AUB, and heterochromatin formation such as Heterochromatin Protein (HP1), contribute to the maintenance of telomere length in Drosophila germline cells by regulating the frequency of HeT-A and TART retrotranspositions (Savitsky et al., 2002; Savitsky et al., 2006) Mutations in spn-E and aub result in an increased retrotransposition and hence, de-repression of HeT-A and TART in the female germline (Savitsky et al., 2006; Vagin et al., 2006), while a mutation in spn-E appears to have no 22 effect on HeT-A expression in the soma (Klenov et al., 2007) In Drosophila somatic cells, Invader4 is de-repressed in the cytosine-5-methyltransferase mutant dnmt2, but is unaffected in spn-E and aub mutants (Phalke et al., 2009) These observations indicate that telomere integrity is differentially maintained in the germline and soma, namely by RNAi-based regulation in the germline and by DNA methylation-dependent maintenance in the soma 1.3.3.2 Female fertility control and hybrid dysgenesis in D melanogaster In D melanogaster, when wild-caught females are crossed to laboratory-kept males, the F1 progenies are sterile, whereas the reciprocal intercrosses yield F1 progenies that are fertile (Figure 1.3.4, (Bregliano et al., 1980; Bucheton, 1990; Kidwell and Kidwell, 1977) This phenomenon is known as hybrid dysgenesis, which is defined as the nonreciprocal inheritance of aberrant traits such as sterility, mutation, and male recombination, in the F1 hybrids (Kidwell and Kidwell, 1977) There are two forms of hybrid dysgeneses, namely inducer-reactive (I-R) and paternalmaternal (P-M) types that manifest in the dysgenic F1 progenies due to the mobilisation of I-element retroelement and P-element DNA transposon, respectively (Brennecke et al., 2008) Active I-element and P-element loci, from which piRNAs are derived, are absent in wild-caught females (R or M type) but present in the laboratory-kept females (I or P type) Hence, F1 progenies arising from intercrosses between wild-caught females and laboratory-kept males lack the inheritance of maternal piRNAs (Brennecke et al., 2008) The lack of maternal piRNAs as a starting material compromises piRNA biogenesis via 23 the feed-forward amplification loop As a consequence, transposition of I-element and Pelement occur, leading to female sterility (Brennecke et al., 2008) Figure 1.3.4 I-R hybrid dysgenesis in D melanogaster An intercross between wildcaught females (R type, green) and laboratory-kept males (I type, red) give rise to sterile, dysgenic F1 progenies The reciprocal cross yields fertile, non-dysgenic F1 progenies 1.3.3.3 Male fertility control in D melanogaster Meiosis and spermatogenesis in male D melanogaster are regulated by a set of paralogous genes: X-linked ste and Y-linked tandem repeats suppressor-of-ste [su(ste)] (Balakireva et al., 1992; Livak, 1984; Palumbo et al., 1994; Tulin et al., 1997) su(ste) repeats contain a ste-like region that is 90% identical to ste and each repeat harbours a 1360 (or hoppel) transposon insertion (Balakireva et al., 1992) su(ste) is transcribed 24 bidirectionally to generate dsRNA that can target ste (Aravin et al., 2001) Mutations in spn-E and aub eliminate the generation of short double-stranded su(ste) RNA, resulting in de-repression of ste and translation of STE protein (Aravin et al., 2004; Aravin et al., 2001; Kotelnikov et al., 2009) An upregulation of STE expression promotes the accumulation of crystalline aggregates in the primary spermatocytes, leading to male sterility 1.4 Post-transcriptional gene silencing and Processing bodies The contribution of several nuage components to the repression of retroelements predicts that silencing is compartmentalised in the nuage Indeed, the mouse homologue of the nuage, chromatoid body, is reported to co-localise with miRNAs let-7 and miR-21 (Beaudoin et al., 2008; Kotaja et al., 2006) In addition, components of processing body (P-body) that are known to participate in mRNA degradation and translational silencing, Decapping Protein 1a (DCP1a) and Glycine-tryptophan protein of 182kDa (GW182), are also co-localised with the chromatoid body In C elegans, P-body components Protein associated with Topo II related family member (PATR-1), Chemokine (C-C motif) receptor associated factor family member (CCF-1), Decapping protein (DCAP2), and Conserved germline helicase family member (CGH-1), overlap with P-granules (a counterpart of the nuage) in germline blastomeres (Gallo et al., 2008; Lall et al., 2005) These findings therefore suggest that the nuage “collaborates” with P-bodies in mediating silencing 25 1.4.1 Processing bodies P-bodies are discrete cytoplasmic foci, often described as the accumulation site for RNP complexes that participate in mRNA degradation and translational repression in both somatic and germline cells (Lin et al., 2008), reviewed in (Eulalio et al., 2007; Parker and Sheth, 2007) The 5’-to-3’ exoribonuclease XRN1 [also known as Pacman (PCM) in D melanogaster] was the first molecule to be reported to exhibit localisation to cytoplasmic granular foci in mice (Bashkirov et al., 1997) However, little effort was made to understand these unique XRN1 foci until the decapping enzyme DCP2 and its cofactors were discovered in both mammalian and yeast cells (Cougot et al., 2004; Ingelfinger et al., 2002; Lykke-Andersen, 2002; Sheth and Parker, 2003) Subsequently, an increasing number of components were identified as constituents of P-bodies and these include proteins involved in mRNA degradation, translational repression and RNAi 1.4.2 mRNA degradation Cellular mRNA turnover is mediated by conventional mRNA degradation enzymes, such as the decapping enzymes, deadenylases, and exoribonucleases Eukaryotic mRNA degradation involves two predominant pathways (Figure 1.4.1), namely the 5’ and 3’ mRNA decay pathways Both pathways require deadenylation, that is the removal of the poly(A) tail, by the Chromatin assembly factor 1- Chemokine (C-C motif) receptor NOT (CAF1-CCR4-NOT) complex as the initiating step (Cougot et al., 2004; Sheth and Parker, 2003; Temme et al., 2004) 26 Figure 1.4.1 Schematic diagram of bulk mRNA degradation in eukaryotic cells In eukaryotic cells, mRNA is degraded by two alternative pathways, both of which require deadenylation The first initiating step of mRNA degradation involves the removal of the poly(A) tail by the CAF1/CCR4/NOT deadenylase complex Subsequently, the decapping enzymes DCP1/2, together with the co-factors such as Dhh1p (also known as Me31B in D melanogaster), remove the cap structure and the 5’-to-3’ exoribonuclease XRN1 (also known as PCM in D melanogaster) catalyses the digestion from the 5’end The alternative mRNA degradation pathway involves the exonucleolytic digestion from the 3’ end by the SKI/exosome complex (adapted from Eulalio et al., 2007) In the 5’ mRNA decay pathway, the decapping enzymes DCP1/2 first remove the cap structure (Behm-Ansmant et al., 2006; Ding et al., 2005; Eulalio et al., 2007; Ingelfinger et al., 2002; Lykke-Andersen, 2002; Sheth and Parker, 2003) and the 5’-to-3’ exoribonuclease XRN1 digests the decapped transcript from the 5’end (Bashkirov et al., 1997; Chernukhin et al., 2001; Ingelfinger et al., 2002; Sheth and Parker, 2003; Zabolotskaya et al., 2008) On the other hand, the 3’ mRNA decay pathway involves 3’27 to-5’ exoribonucleolytic digestion of the transcript by the SKI/exosome complex, followed by the removal of the cap structure (Houseley et al., 2006; Orban and Izaurralde, 2005) A majority of these enzymes and RNA binding proteins such as GW182, are reported to localise to the P-bodies (reviewed in Anderson, 2005; Eulalio et al., 2007; Parker and Sheth, 2007), suggesting P-bodies as major sites for mRNA decay 1.4.3 RNA interference Studies have implicated RISC-mediated mRNA degradation at the P-bodies, where components of the RISCs are shown to localise to these cytoplasmic sites in human cultured cells (Jagannath and Wood, 2008; Liu et al., 2005b; Sen and Blau, 2005 ) Furthermore, biochemical analyses have demonstrated that the P-body components GW182 and DCP1 interact with the RISC components AGO1 and AGO2 in mammalian cells (Behm-Ansmant et al., 2006; Liu et al., 2005a; Liu et al., 2005b), and that the C elegans homologue of GW182, Acyl-CoA carboxylase insensitive (AIN-1), interacts with a putative AGO family protein, Asparagine-linked glycosylation (ALG-1) (Ding et al., 2005) These observations imply that small RNA-mediated mRNA degradation and/or translational repression take place post-transcriptionally in the P-bodies In the case of RISC-mediated mRNA degradation, siRNA- and plant miRNA-bound AGO proteins probably associate with the target mRNAs (Figure 1.4.2) Following which, AGO proteins promote endoribonucleolytic cleavage(s) of the mRNAs by means of their slicer activities to generate 5’ and 3’ decay intermediates The decay intermediates are then removed by the mRNA degradation enzymes such as XRN1 and SKI/exosome 28 complexes via their exoribonucleolytic activities (reviewed in Bartel, 2004; Fillpowicz, 2005; Orban and Izaurralde, 2005; Souret et al., 2004) Figure 1.4.2 Schematic diagram showing siRNA-mediated gene silencing siRNAbound AGO protein promotes endoribonucleolytic cleavage of the target mRNA to generate the 5’ and 3’ decay intermediates The 5’ decay intermediate is digested by the 5’-to-3’ exoribonuclease XRN1, while the 3’ decay intermediate is removed by the SKI/exosome complex (adapted from (Eulalio et al., 2007) 1.5 RNA silencing and endosomal trafficking Besides P-bodies, mRNAs and miRNAs are reported to be present in the exosomes, which are one of the vesicular compartments of the endosomal pathway (Skog et al., 2008; Valadi et al., 2007) This observation suggests a connection between RNA silencing and trafficking, which is further supported by evidences that the formation and turnover of RISCs are linked to and modulated by the endosomal pathway (Figure 1.5.1; Gibbings et al., 2009; Lee et al., 2009) 29 Figure 1.5.1 RISC assembly and turnover occur at endosomes Blocking the Endosomal Sorting Complex Required for Transport (ESCRT)-dependent formation of MVBs inhibits RISC activity, while blocking the fusion of MVBs with lysosomes stimulates RISC activity Hence, ESCRT-dependent removal of GW182 by fusion with lysosomes may promote the assembly of miRNA-RISCs for silencing (adapted from Siomi and Siomi, 2009) GW, GW182 The endosomal pathway is defined by the internalisation and sorting of cargo proteins to different compartments within a cell Upon internalisation, cargo proteins are ubiquitinylated and sorted by the Endosomal Sorting Complex Required for Transport (ESCRT) complex to late endosomes or multivesicular bodies (MVBs) MVBs either enter the exocytic pathway to secrete exosomes that release the vesicular contents into the extracellular space, or mature further into lysosomes that contain hydrolytic enzymes for protein turnover (Figure 1.5.2; reviewed in Siomi and Siomi, 2009) 30 Figure 1.5.2 Schematic diagram depicting endosomal trafficking in a cell Membrane proteins (blue) are internalised by the cell to form vesicles The endocytic vesicles fuse with early endosomes and mature into late endosomes/MVBs MVBs will either enter the exocytic pathway to secrete exosomes that release the vesicular contents into the extracellular space, or mature further into lysosomes that contain hydrolytic enzymes that promote protein turnover GW182 aggregates (GW) and miRNA-bound AGO appear to localise with the late endosomes, constituting a subset of RNPs that is distinct from Pbodies (adapted from Siomi and Siomi, 2009) PB, processing bodies GW, GW182 bodies SG, stress granules Components of the RISCs, AGO1 and AGO2, are found to associate closely with an endosomal marker LAMP1 and an exosome marker CD63 In ESCRT RNAi mutant, the sorting of ubiquitinylated cargo proteins to MVBs is compromised and miRNA-RISCmediated silencing is downregulated Conversely, when the fusion of MVBs with 31 lysosomes is blocked in Hermansky-Pudlak Syndome (HPS4) mutant, miRNA-RISC loading and silencing are upregulated (Gibbings et al., 2009; Lee et al., 2009) Lee et al (2009) further demonstrates that small RNA loading and silencing are impaired in the Drosophila ubiquitin ligase mutant dFBX011, indicating that protein ubiquitinylation is required to facilitate the loading of small RNAs onto RISCs Taken together, these new findings demonstrate that RNA silencing and endosomal trafficking are linked, where components of the endosomal pathway aid in small RNA loading and RISC turnover 1.6 Thesis overview My thesis aims to understand the cell biology and germline contributions of the nuage, an evoluntionarily-conserved organelle in the animal kingdom Using D melanogaster ovary as the model system, I describe a newly-identified nuage component gene krimper (krimp) Characterisation of this gene indicates that krimp regulates oocyte polarity, oocyte meiotic progression, and oskar mRNA translation KRIMP, as well as other nuage components such as VAS, AUB, and MAEL, participates in retroelement silencing by regulating sufficient piRNA production Nuage that is found in the cytoplasm overlaps retroelement transcripts, piRNAs, and proteins involved in mRNA degradation These cytplasmic bodies are termed pi-bodies, which possibly represent sites at which RNAi and mRNA degradation proteins gather to eliminate retroelement transcripts The assembly of pi-bodies is dependent on piRNAs and appears to correlate with retroelement silencing By analysing the decay and/or stabilisation of an artificial retroelement mRNA in vivo, I show that retroelement silencing is in part post-transcriptional and piRNA- 32 dependent Lastly, mRNA degradation proteins also contribute to retroelement silencing, either in a piRNA-dependent or -independent manner 33 ... of processing body (P-body) that are known to participate in mRNA degradation and translational silencing, Decapping Protein 1a (DCP 1a) and Glycine-tryptophan protein of 18 2kDa (GW182), are also... et al., 2005b), and that the C elegans homologue of GW182, Acyl-CoA carboxylase insensitive (AIN -1) , interacts with a putative AGO family protein, Asparagine-linked glycosylation (ALG -1) (Ding... small RNA-mediated silencing Indeed, a preliminary study that examined the small RNA profiles of different developmental stages in D melanogaster has identified a unique class of small RNAs that