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

In this thesis, I have described a function for the nuage in mediating post-transcriptional retroelement silencing.. 4.1 Nuage role in post-transcriptional regulation Using the Drosophi

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Figure 3.4.2 Cytoplasmic KRIMP do not overlap with PDI-GFP KRIMP (red)

cytoplasmic foci do 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

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4 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

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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 piRNA-dependent 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

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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

HeT-A 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

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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

post-111

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 GTP-binding 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

112

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 protein 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

multi-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

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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 coiled-coil 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

mouse Piwi, arginine residues are also dimethylated in Drosophila AUB and AGO3

(Kirino et al., 2009) Protein arginine methyltransferase 5 (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

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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 In fact, one of the nuage

components AUB has been implicated in the P-M hybrid dysgenesis, where P-element repression in the euchromatin is sensitive to aub mutation and appears to be mediated by

RNA molecules that are derived from the heterochromatin (Reiss et al., 2004; Simmons

et al., 2007) Unequivocally, a recent study by Brennecke et al (2008) demonstrates that

the repression of P-element in the female germline of D melanogaster is mediated by

maternally-inherited piRNAs, suggesting the involvement of small RNAs in the silencing

of DNA transposons

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4.5.2 Does the nuage function in the soma?

Two complementary works that are published recently have provided evidences of an ovarian somatic piRNA pathway that functions in a ping pong cycle and AGO3-independent manner to generate somatic piRNAs (Li et al., 2009; Malone et al., 2009) Among all the nuage components that the authors have examined, only ZUC appears to

function in the soma to produce flamenco-derived piRNAs Other nuage components

such as SPN-E, VAS, AUB, KRIMP, and ARMI, in turn, contribute differentially to the biogenesis of germline piRNAs derived from the 42AB cluster (Malone et al., 2009) Besides, the nuage/RNAi machinery SPN-E and AUB have been reported to exert

functions on heterochromatin silencing in the eye and salivary glands of D melanogaster

(Pal-Bhadra et al., 2004)

A preliminary finding in our laboratory has indicated the presence of VAS, KRIMP, and MAEL transcripts in the wild-type adult heads (unpublished) This predicts the nuage contribution in the nervous tissues, possibly in the silencing of retroelements Indeed, the

non-LTR retroelement L1 is reported to manifest in the human neural progenitor cells (Coufal et al., 2009) In D melanogaster, the P-body proteins Me31B, PCM, and dDCP1

are expressed in cultured motor neurons derived from the larval brain (Barbee et al., 2006)

Taken together, somatic tissues appear to utilise similar forms of silencing machinery Hence, it will be exciting to discern the assembly of pi-body-like RNP complexes, as well

as probable nuage somatic function(s) with relation to retroelement expression in a

117 piRNA- and P-body-dependent manner Finally, it will also be interesting to elucidate the importance of retroelement silencing in the progenitor cells of multiple tissues

118

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