Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 1

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 ...... Besides localising to th

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:

1 My supervisor, Dr Toshie Kai, for her conscientious guidance and supervision throughout my graduate studies

2 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

3 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

4 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especially to H Han (McGill University, Montreal, Quebec, Canada) for her unpublished anti-AUB

5 TLL facilities, Media prep, IT department, and Microscopy department, for their support in the flyfood preparation and image acquisition

6 My spouse, You Keat Chia, for his boundless love and encouragement during my hustling work hours

7 My TLL friends, Woei Chang Liew, Alex Chang, and Kaichen Chang, for sharing my laughter and troubles

8 My climbing and diving friends, for bringing joy to my graduate student life

9 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  Telomere maintenance 21 

1.3.3.2  Female fertility control and hybrid dysgenesis in D melanogaster 23 

1.3.3.3  Male fertility control in D melanogaster 24 

1.4  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  Thesis overview 32 

2  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  Microscopy and image processing 48 

2.4  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  In vitro co-IP 53 

2.4.5  Northern analyses of transcripts and piRNAs 53 

2.5  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  Primers 56 

3  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  piRNA-mediated retroelement silencing is post-transcriptional 97 

3.4  Nuage and endosomal trafficking 104 

4  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 

I

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

de-repressed 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

II

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 transcriptional retroelement silencing HeT-A mRNA is de-repressed in mRNA

post-degradation mutants, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates

III

Lists of Tables

Table I Summary of the nuage components and their functions 11 

Table II Antibodies for immunohistochemistry 46 

Table III Antibodies for immunoblotting 52 

Table IV Primer sequences 56 

IV

List of Figures

Figure 1.1 Electron micrographs of the nuage 3

Figure 1.1.1 Schematic diagrams of the D melanogaster ovary 5 

Figure 1.1.2 Asymmetric localisation of oocyte mRNAs in a D melanogaster stage 7 egg chamber 6 

Figure 1.1.3 Establishment of polarity by signaling relay between the soma and germline 7 

Figure 1.1.4 Germline formation in D melanogaster ovary 8 

Figure 1.3.1 Schematic diagrams depicting biogenesis of small RNA biogenesis and post-transcriptional silencing 18 

Figure 1.3.2 Ping pong model for piRNA biogenesis 20 

Figure 1.3.3 Schematic representation of chromosome 3R telomere-associated sequences in D melanogaster 22 

Figure 1.3.4 I-R hybrid dysgenesis in D melanogaster 24 

Figure 1.4.1 Schematic diagram of bulk mRNA degradation in eukaryotic cells 27 

Figure 1.4.2 Schematic diagram showing siRNA-mediated gene silencing 29 

Figure 1.5.1 RISC assembly and turnover occur at endosomes 30 

Figure 1.5.2 Schematic diagram depicting endosomal trafficking in a cell 31 

Figure 3.1.1 KRIMP is a nuage component 60 

Figure 3.1.2 Subcellular localisation of KRIMP in D melanogaster germline cells 61 

Figure 3.1.3 Homologues of KRIMP are identified in the Drosophilidae family 62 

Figure 3.1.4 krimp f06583 is a loss-of-function allele 63 

Figure 3.1.5 krimp mutant exhibits meiotic progression defects 65 

Figure 3.1.6 krimp mutant exhibits oocyte polarity defects 66 

V

Figure 3.1.7 Nuage component mutants exhibit precocious osk translation 68 

Figure 3.1.8 KRIMP-Venus(YFP) localises to the perinuclear nuage in the ovary 69 

Figure 3.1.9 UASp-krimp-venus transgene fully rescues krimp mutant defects 70 

Figure 3.1.10 Nuage components interact genetically with one another 72 

Figure 3.1.11 KRIMP interacts directly with AUB, CUFF, and AGO3 73 

Figure 3.1.12 KRIMP interacts with AGO3 in vivo 74 

Figure 3.1.13 Retroelements are de-repressed in krimp mutant 75 

Figure 3.1.14 Schematic drawing depicting KRIMP transgene variants 76 

Figure 3.1.15 KRIMP domains display distinct functions 77 

Figure 3.1.16 KRIMP-NT and CT do not interact with AGO3 in vitro 79 

Figure 3.1.17 KRIMP-NT restores retroelement repression 80 

Figure 3.2.1 LINEs are de-repressed in the nuage component mutants 82 

Figure 3.2.2 Nuage localisation is unaffected in the conventional dicing enzyme mutants dcr-1 and dcr-2 84 

Figure 3.2.3 Production of piRNAs is defective in the nuage component mutants 85 

Figure 3.3.1 Nuage/piRNA pathway components exhibit both perinuclear and cytoplasmic foci 86 

Figure 3.3.2 Nuage cytoplasmic foci overlap mRNA degradation proteins of the P-bodies 88 

Figure 3.3.3 The association of the cytoplasmic nuage and P-body foci is highly dynamic 89 

Figure 3.3.4 Retroelement transcripts co-localise with nuage cytoplasmic foci and mRNA degradation proteins 91 

Figure 3.3.5 Antisense piRNAs co-localise with GFP-HeT-A bodies 93 

Figure 3.3.6 Assembly of the pi-bodies is piRNA-dependent 94 

VI

Figure 3.3.7 Deadenylation is unaffected in aub mutant LM-PAT assay of cycB 95 

Figure 3.3.8 Decapping is unaffected in aub mutant 96 

Figure 3.3.9 The protein expression of mRNA degradation proteins is unaffected in piRNA pathway mutants 97 

Figure 3.3.10 Post-transcriptional retroelement silencing is piRNA-dependent 98 

Figure 3.3.11 Full-length retroelement transcripts accumulate in the piRNA pathway and deadenylase mutants in vivo 99 

Figure 3.3.12 HeT-A is de-repressed in dcp1 and ski3 mutants 101 

Figure 3.3.13 piRNA production is unaffected in the mRNA degradation mutants 102 

Figure 3.3.14 Nuage localisation is unaffected in the mRNA degradation mutants 103 

Figure 3.4.1 Cytoplasmic nuage is tethered to ER/endosomal compartments 105 

Figure 3.4.2 Cytoplasmic KRIMP do not overlap with PDI-GFP 106 

VII

List of Abbreviations

Ag antigen

AGO Argonaute

CB cytoblast

DIG digoxygenin

FISH fluorescence in situ hybridisation

GFP Green Fluorescence Protein

RISC RNA-induced silencing complex

RNP ribonucleoprotein

YFP Yellow fluorescence protein

It has been appreciated since the 18th century that the continuity of germ cells depends

on cytoplasmic germinal determinants that are passed on to the daughter cells A studied germline determinant is the pole plasm (or germ plasm), which is a specialised RNA-rich region located at the posterior end of the oocyte (Mahowald, 1971a) The pole plasm comprises of electron-dense, membraneless granules (polar granules) that contribute to primordial germ cell (PGC) formation and abdominal patterning (Sander, 1976) Interestingly, the pole plasm is thought to share resemblance with another evolutionarily conserved feature of the germline, known as the nuage (Eddy, 1975; Hay

well-et al., 1988; Lasko and Ashburner, 1990) The nuage, which means “cloud” in French, is commonly observed in over 80 animal germlines across eight phyla (Eddy, 1975; Hay et

al., 1988; Lasko and Ashburner, 1990) In mouse testes and Caenorhabditis elegans

persists throughout oogenesis in the adult germline cells RNase and protease experiments have shown that the nuage is enriched with RNAs and proteins (Mahowald,

1968; Mahowald, 1971a; Mahowald, 1971b)

Despite the wealth of both descriptive and structural information collected over the past two centuries, the cell biology of the nuage still remains poorly understood One outstanding question is how the nuage, as a characteristic feature of germ cells, contributes to germline development

3

Figure 1.1 Electron micrographs of the nuage (a) Two gonadal germ cell nuclei in C

elegans P-granules (Pg, black arrows) form electron-dense amorphous structures around

the nuclei (nu, adapted from Pitt et al., 2000) (b) Wild-type stage 6 nurse cell nucleus in

D melanogaster ovary The nuage (p) forms an electron-dense structure on the cytoplasmic face of the nurse cell nuclear envelope in D melanogaster (adapted from

Liang et al., 1994) (c) Seminiferous tubule segments in mouse spermatids The

chromatoid body is an irregular network of dense filaments (white arrows, adapted from Kotaja and Sassone, 2007)

Using D melanogaster ovary as the model system, I show that the conservation of the

nuage throughout evolution is attributed to its importance in governing genome integrity through the repression of retroelements, a class of mobile genetic elements This is of significance in the germline since the random transpositional nature of retroelements may compromise genome fidelity Compromising genome integrity is undesirable for germ

a

4

cells since they must ensure accurate transmission of genetic material to the next generation I also demonstrate that retroelement repression is mediated by a unique class

of small RNAs, known as the P-element-induced wimpy testes (Piwi)-interacting RNAs

(piRNAs) and is in part post-transcriptional In addition, nuage that is present in the cytoplasm appears to be associated with proteins involved in mRNA degradation, retroelement transcripts, and piRNAs, suggesting the involvement of a ribonucleoprotein complex (RNP) in retroelement silencing

1.1 Drosophila melanogaster germline as a model system

The ease of culture, well-established genetics, simple gonadal architecture, and

availability of mutant alleles have rendered D melanogaster a popular model organism to

study the cell biology of the nuage Moreover, the fruitfly has a short life cycle of approximately ten days and each female fly can lay as many as 200 eggs per day

Each female fly has two ovaries, each comprising of 16-20 ovarioles that represent individual assembly lines of egg chambers (Figure 1.1.1a) At the tip of each ovariole is the germarium, where 2-3 germline stem cells (GSCs) reside Each GSC divides asymmetrically to give rise to a cystoblast (CB), which undergoes four mitotic cell divisions with incomplete cytokinesis to form a 16-cell cyst (Figure 1.1.1b) The 16-cell cyst is encapsulated by a sheath of follicle cells upon emergence from the germarium, forming an egg chamber Among these 16 cells, one cell differentiates to become the oocyte, while the remaining 15 cells take up the role of nurse cells The constant provision of nutrients (“nursing”) by the nurse cells promotes the enlargement of the

5

oocyte until stage 14 of oogenesis when a mature egg chamber forms and is ready for fertilisation

Figure 1.1.1 Schematic diagrams of the D melanogaster ovary (a) An ovariole,

showing a germarium at the anterior, and egg chambers with numbers indicating the stages of oogenesis (adapted from Keyes and Spradling, 1997) Each egg chamber comprises of 16 germline cells (15 nurse cells and 1 oocyte), encapsulated by a sheath of

follicle cells (b) Cyst differentiation One CB undergoes four mitotic cell divisions with

incomplete cytokinesis to form a 16-cell cyst that is interconnected by the ring canals (green) The cytoskeletal organelle fusome (red) is inherited by the cysts (adapted from Kai and Spradling, 2004)

The formation of a mature egg chamber is a highly complex process that requires proper specification of oocyte polarity, as well as the accumulation of RNA-rich granules known

as polar granules (Hegner, 1914) The establishment of oocyte polarity is fundamental to body axis specification and is thus critical to promote proper cellular development in the adult fly Oocyte polarity specification depends on the localisation of several mRNAs

such as bicoid (bcd), oskar (osk), nanos (nos), and gurken (grk; reviewed in Becalska and Gavis, 2009; Steinhauer and Kalderon, 2006) bcd and osk mRNAs are asymmetrically

localised to the anterior and posterior of the oocyte to establish anterior/posterior (A/P)

b

a

6

polarity (Berleth et al., 1988; Ephrussi and Lehmann, 1991; Kim-Ha et al., 1991;

Nusslein-Volhard et al., 1987), while the localisation of grk mRNA to the antero-dorsal

region specifies dorsal/ventral (D/V) polarity (Figure 1.1.2; Neuman-Silberberg and

Schupbach, 1993) nos mRNA localises to the posterior of the oocyte during the late stages of oogenesis following osk translation and plays important roles in the germline

and abdominal development (Lehmann and Nusslein-Volhard, 1986; Wang et al., 1994)

Figure 1.1.2 Asymmetric localisation of oocyte mRNAs in a D melanogaster stage 7 egg chamber bcd and osk mRNAs localise to the anterior and posterior regions to

specify A/P polarity, while grk mRNA localises to the antero-dorsal region to specify D/V polarity nos mRNA is not localised at this stage GRK signals to the overlying

follicle cells to determine the future dorsal region (adapted from Morris and Lehmann, 1999)

The specification of oocyte polarity has been shown to be microtubule-dependent (Lipshitz and Smibert, 2000; St Johnston, 1995) During early stages of oogenesis, the nurse cells and oocyte are approximately the same size and a single microtubule

7

organising centre (MTOC) extends from the oocyte into the nurse cells (Figure 1.1.3a)

At stages 6-7 of oogenesis, signals from the overlying follicle cells to the oocyte trigger the reorganisation of the microtubules to form a polarised network (Figure 1.1.3b-c, reviewed in (Eeden and St Johnston, 1999) This, in turn, promotes the migration of the oocyte nucleus to the anterior cortex, which specifies the future dorsal region and

localisation of the grk mRNA Similarly, the polarised microtubule network determines the asymmetric localisation of bcd and osk mRNAs to the anterior and posterior regions

in the oocyte, respectively (Figure 1.1.3d)

Figure 1.1.3 Establishment of polarity by signaling relay between the soma and germline (a) During stages 2-6 of oogenesis, one MTOC originates from the oocyte

nucleus and the positive ends of the microtubules (red solid lines) extend into the nurse

cells (b-c) Signals from the overlying follicle cells (purple) promote the disassembly (red

dash lines) and reorganisation of microtubules (red solid lines) to form a polarised

network (d) grk mRNA (green) localises to the antero-dorsal region of the oocyte, which

specifies the future dorsal axis osk (yellow) and bcd (blue) mRNAs localise to the

posterior and anterior regions of the oocyte to specify the A/P axis (adapted from Steinhauer and Kalderon, 2006)

Besides establishing proper oocyte polarity, the accumulation of polar granules at the pole plasm is essential to ensure fertility and eventually, perpetuation of an organism (Figure 1.1.4)

8

Figure 1.1.4 Germline formation in D melanogaster ovary In the early egg chamber,

the oocyte is nursed by cytoplasmic streaming of RNAs and proteins In the stage 10 egg chamber, the ooctye nucleus migrates to the posterior region of the oocyte and polar granules accumulate The oocyte continues to enlarge, while the nurse cells start to degenerate By stage 14, the pole plasm forms at the posterior end of the egg chamber At the syncytial blastoderm stage, pole cells (future germline cells) form after the migration

of the nuclei to the cell periphery Cellularisation then begins (adapted from Saffman and Lasko, 1999)

Intriguingly, reports have suggested that the nuage closely resembles the pole plasm since they share many characteristics, such as structure and morphology, localisation to the