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A centromeric RNA-associated protein complex affects germ line development in Drosophila melanogaster

In many metazoans, centromeres are embedded in large blocks of highly repetitive (peri-) centromeric heterochromatin from which non-coding RNAs emanate that have been assigned diverse functions in different species. However, little is known about their functional details or regulation. The pericentromere of the X chromosome in Drosophila melanogaster contains a multi mega-base array of the 359 bp satellite repeats from the 1.688 family, which is transcribed into a lncRNA (SAT III RNA). We performed a SAT III RNA pulldown assay and identified a SAT III RNA-associated complex of four previously uncharacterized proteins and show that they affect germline development. These factors not only interact with each other and with SAT III RNA but also co-regulate each other. RNAi depletion of any of the factors leads to severe defects in the developing germline and sterility. Moreover, we show that the complex plays a crucial role in SAT III RNA repression, as RNAi depletion of the factors leads to a drastic increase of SAT III RNA levels. Importantly, genetic reduction of SAT III RNA level in the RNAi-depleted flies partially rescued the germ line defects and infertility phenotype. Based on our results we hypothesize that the identified complex functions in the germline to regulate SAT III RNA levels, possibly to offset effects of chromatin remodelling taking place in the developing germline.

Introduction established links to mitosis or centromeric RNAs. The human orthologue of the putative ribosomal-like protein CG13096 (RSL1D1) was identified in a human alpha satellite RNA pulldown (Zhu, Hoong et al., 2018) and the human orthologue of the putative RNA methyltransferase CG12128 (SPOUT1 or CENP-32) was enriched at mitotic spindles and kinetochores, being important for tethering the mitotic spindle to centrosomes and kinetochores (Ohta, Bukowski-Wills et al., 2010, Ohta, Wood et al., 2015. The nucleolar protein CG1234 had been identified to cause shorter mitotic spindles upon knockdown (KD) (Somma, Ceprani et al., 2008), and the human orthologue (NOP2) of the putative RNAmethyltransferase CG8545 was enriched in a human alpha satellite RNA pulldown (Zhu et al., 2018). In addition, CG8545 was co-immunoprecipitated with CENP-A in Drosophila cells (unpublished data from our lab). Similarly, the putative DEAD-box RNA helicase CG32344 was found in a CENP-A pulldown of associated factors in Drosophila S2 cells (Barth, Schade et al., 2015). Generally, DEAD-box helicases have been shown to bind satellite I ncRNAs in humans contributing to accurate chromosome segregation (Nishimura, Cho et al., 2019). Even though CG12128 is an interesting candidate found in both SAT III RNA pulldowns, subsequent experiments showed that it does not share the same phenotypes as the other four candidate proteins and was, therefore, either excluded from our studies or used as a control. The remaining four uncharacterized proteins turned out to be expressed in the gonads and to be important for germline development during subsequent experiments (Fig. Centagon 1-GFP and Centagon 4-GFP flies were heterozygous since homozygous stocks were not viable. The weaker GFP signals in those lines may be attributed to their heterozygosity rather than a lower expression compared to Cent2 and Cent3. Importantly, Centagon protein expression is not limited to the gonads, as GFP signal was also observed in larval brain tissue, larval imaginal discs and fat tissue ( Fig. 3B + C). In the germarium, Centagon proteins are highly expressed in the germ stem cells (GSCs, marked by pMad immunostaining) and first cystoblasts, but undetectable in cells derived from subsequent divisions (Fig. 3D). The Centagon complex is also expressed in the accompanying somatic cells. Only at egg chamber stage 1, expression increases again in the germ cells, indicating a differential Centagon expression pattern during early germ cell divisions.
To confirm that the Centagon complex also binds SAT III RNA in vivo and in ovary, we performed RNA-Immunoprecipitation (RIP) with ovary lysate from the two homozygous lines of the endogenously tagged Centagons, Cent2 and Cent3, and analysed the SAT III RNA levels by qPCR. We detected a significant enrichment of SAT III RNA in the Cent2-IP compared to the control GAPDH mRNA (Fig. 3E + S3E). SAT III RNA was also enriched in the Cent3 RIP but only two out of three repeats showed a strong enrichment ( Fig. 3F + S3E). This result suggests that also in ovaries, the Centagon complex binds SAT III RNA.

The Centagon complex is important for oogenesis.
To study the germ line function of the Centagon complex we next performed knockdown experiments of the Centagon members in ovaries. To do so, we crossed TRiP RNAi fly lines targeting Centagon transcripts with two ovary-specific Gal4 driver lines: Maternal Triple driver (MTD-Gal4) and Mat67.15-Gal4 (Grieder, de Cuevas et al., 2000, Mazzalupo & Cooley, 2006, Staller, Yan et al., 2013. MTD-Gal4 is expressed already in GSCs and Mat67.15-Gal4 starts expression in early egg chambers (Fig. S3C). We dissected the ovaries of the resulting female offspring and observed an obvious size difference between control and all Centagon KD ovaries. In contrast, KD of CG12128, which we used as control, did not affect ovarian development (Fig. S3D). KD of the Centagon members during early germ cell development (with MTD-Gal4) led to more severe phenotypes with strongly underdeveloped ovaries compared to KD at later stages (with Mat67.15-Gal4) suggesting a function during early germ line development. The efficiency of the Mat67.15-induced KD was monitored by qPCR ( Fig.   4A). Not only were the mRNA levels of the Centagon members significantly reduced when depleted by RNAi, upon KD of one member, levels of the other three Centagon members were significantly upregulated, emphasizing the existence of a functional complex and suggesting co-regulation and interdependence of the Centagon complex components. No qPCR data for the MTD-induced KD efficiency is available because there was too little tissue left after knockdown. However, since the phenotype was consistent with the one from the Mat67.15 induced KD, we are confident that the KD was also highly efficient.
Immunofluorescent imaging showed that in the early MTD-induced Centagon KD ovaries, almost no vasa-positive germ cells were left in the germaria and only very few immature egg chambers were observed (Fig. 4B, white arrows). Interestingly, this was not due to a loss of GSCs, since many pMad-positive cells were left in the stem-cell niche. However, we observed aberrant pMad expression at the germ cell cyst stage (red arrows), which was significantly higher in the Centagon 1 KD ovaries compared to the control (Fig. 4C). This result indicates a possible differentiation defect of germ cells upon Centagon depletion with cells maintaining a stem cell-like potential longer than under control conditions. When the Centagon members were knocked down in early egg chambers (with Mat67.15-Gal4), egg chambers ceased to develop and displayed abnormally fragmented nurse cell nuclei that were reminiscent of the 5-blob phenotype (Fig. 4D + E) (Dej & Spradling, 1999). In these ovarioles, most egg chambers degenerated before reaching a mature egg stage. The phenotype was strongest in young females (2-4 days). In older females (5-7 days), nurse cells were less fragmented and more egg chambers developed (right panel), indicating some adjustment of the phenotype with age. However, the eggs laid by both young and old females were smaller with no dorsal filaments, which we categorized into three groups: normal, medium (slightly deformed) and small (without dorsal filaments) (Fig. 4F). A survival assay showed that KD of the Centagon members in the ovary leads up to a 20-fold reduction of eggs laid overnight (KD of Cent2) with most of them being deformed. Unsurprisingly, there is a clear correlation between egg phenotype severity and the hatching rate (survival rate) of these eggs after 24 hours (Fig. 4G). We concluded that the Centagon complex is both important during early germ cell divisions and for egg chamber development.
As the Centagon complex is also expressed in somatic ovary cells and germ cells of the testis, we also conducted KD experiments in these cell types. Depletion in the ovary follicle cells resulted in compound egg chambers, follicle cell gaps and, in case of depletion in follicle stem cells, to loss of the entire ovary ( Fig. S4A + B). It is important to note that even though the expression profiles for Centagon components in adult testis are not as high in the FlyAtlas Anatomical Expression (Chintapalli, Wang et al., 2007) and modENCODE Tissue Expression Data sets (Brown et al., 2014) compared to adult ovaries, we detected strong GFP signals in the developing testis of endogenously tagged flies and a strong male germ line phenotype upon depletion. Centagon depletion in the testis led to smaller, deformed testes with a misplaced hub and no sperm formation (Fig. S4C). Therefore, we concluded that the Centagon complex is important for the development of somatic and germ cell development in the male and female germ line of Drosophila melanogaster.

SAT III RNA levels are repressed by the Centagon complex in ovaries
As the Centagon complex was initially identified as a SAT III RNA-associated complex, we next investigated if the depletion of Centagon members has an effect on SAT III RNA levels and localization. First, we performed SAT III RNA FISH in ovaries to determine the SAT III RNA localization in w 1118 control flies (Fig. 5A). In w 1118 ovarioles the SAT III RNA localized in one to several foci in nurse cell nuclei as well as inside the oocyte nucleus (see close-up). To test the specificity of the FISH signal, we used the Zhr 1 fly line which carries a chromosomal translocation that removed the majority of the pericentric SAT III DNA region (Ferri et al., 2009, Sawamura, Yamamoto et al., 1993. We tested both SAT III sense and antisense RNA FISH, but observed a more specific signal with the antisense probes: Although the signals of SAT III sense and antisense probes showed similar localizations in ovarioles, some SAT III sense RNA FISH signal was also detectable in Zhr 1 flies (Fig. S5A), indicating that there are a few SAT III repeats remaining in Zhr 1 flies or that the SAT III sense probe may also detect other satellite repeats. Therefore, further FISH experiments were conducted with SAT III antisense probes. Indeed, Zhr 1 flies did not show any of the SAT III RNA FISH foci that we observed in w 1118 ovarioles (Fig. 5A). To ensure that the observed signal in control flies was indeed RNA and not DNA, which are both mostly lacking in Zhr 1 flies, we treated the ovaries with RNase A. All SAT III RNA foci in w 1118 ovaries disappear after treatment with RNase A, indicating the specific hybridization of SAT III probes to RNA (Fig. S5B). Importantly, the SAT III RNA FISH signal increased drastically in Mat67.15-induced Centagon KD ovaries ( Fig. 5A + B). Although the Centagon members were mostly identified in the SAT III sense RNA pulldown, the RNA FISH experiments show that SAT III sense and antisense transcripts are both upregulated after Centagon KD. This increase was further confirmed by qPCR, where we measured an up to 50fold increase of SAT III RNA levels in Centagon KD ovaries compared to the control (Fig. 5C).
Furthermore, the spatial resolution of the SAT III RNA FISH revealed that SAT III RNA levels were specifically increased in egg chambers with fragmented nurse cells. To quantify this effect, we measured SAT III RNA levels in KD ovaries of 5-7-day old females with both fragmented and normal nurse cell chromatin and found a highly significant increase of SAT III RNA signals in nurse cells with fragmented chromatin ( with Centagon 1 depletion and the Zhr 1 X chromosomes laid as many eggs as the control w 1118 females (Fig. 6C). However, many of these eggs were deformed and had a lower survival rate. It should be noted that the KD efficiency of Centagon 1 was slightly lower in Zhr 1 flies ( Fig. 6D), which could be partially responsible for the reduced phenotype we observed.
Additionally, SAT III RNA levels were assessed by qPCR (Fig. 6E), which indeed remained low in Zhr 1 flies. We concluded that the effect observed after Centagon KD is at least partially caused by high SAT III RNA levels resulting from the lack of repressive function of the Centagon complex. In conclusion, we identified a complex that regulates (peri)centromeric SAT III transcript levels, which is essential for normal somatic and germ cell development in the female gonad and may have additional functions in the male germline and potentially other tissues where we detected high levels of the Centagon complex.

Discussion
There are longstanding questions as to what extent repetitive regions of the genome are functionally relevant and whether transcription or the transcripts themselves are important for genome regulation and cellular functions. In addition, how these transcripts are regulated is not well understood. We focused on the long non-coding RNA SAT III from the repetitive (peri-)centromeric region of the X chromosome of Drosophila melanogaster and show that SAT III RNA is regulated by a novel RNA-binding complex of at least four different proteins that we termed 'Centromeric Transcript-Associated Gonadal' (Centagon) complex. We show here that the Centagon complex represses SAT III RNA levels in the female gonad and influences germ line development and fertility.
Previously, SAT III RNA has been shown to promote correct chromosome segregation during mitosis since depletion of SAT III RNA or the genomic locus causes segregation defects in Drosophila cells and embryos (Rosic et al., 2014). Here we show that elevated SAT III RNA levels are also detrimental for gonadal cells in vivo: The Centagon complex controls SAT III RNA levels in the Drosophila gonad and depletion of complex members resulted in an up to 50-fold increase of SAT III transcript levels accompanied by massive defects in egg chamber maturation. Importantly, reducing the SAT III RNA level in Centagon-depleted ovaries partially rescues the phenotypes. We therefore conclude that accurate levels of SAT III RNA are fundamental to cell survival and development, and tightly regulated. Interestingly, the Centagon complex is important in both actively dividing early germ cells and in postmitotic nurse cells during egg chamber maturation (Fig. 4) indicating an effect of SAT III RNA upregulation outside of mitosis. Depletion of the Centagon complex in the germarium during early germ cell divisions led to empty ovarioles and aberrant expression of the germ stem cell marker pMad. This indicates that the Centagon complex is important for correct germ cell differentiation. Interestingly, the Centagon proteins were amongst 646 factors from a transcriptome-wide RNAi screen that aimed to identify networks controlling stem cell selfrenewal and differentiation in female gonads (Sanchez, Teixeira et al., 2016).
Because the depletion of Centagon components in S2 cells did not result in obvious defects, its role in regulating SAT III levels might be restricted to certain stages and tissues during development as suggested by the high expression levels of endogenously tagged Centagon components in larval tissue. Centagon levels seem particularly high in tissues that undergo changes in their developmental potential such as the germline and imaginal discs.
This may include changes in heterochromatin and strict regulation of transcripts from repetitive elements by the nucleolar Centagon complex. This is supported by previous reports that the localization of centromeres to an intact nucleolus is important for heterochromatin formation and transcriptional silencing of repetitive elements in Drosophila and human cells (Bury et al., 2020, Padeken, Mendiburo et al., 2013.
It is important to note that the Centagon complex components have been implicated in ribosome biogenesis: The yeast orthologue of Cent1 is involved in 60S ribosomal subunit maturation (Milkereit, Gadal et al., 2001), while Cent2 as a putative RNA methyltransferase may be involved in rRNA maturation as has been documented for the nucleolar RNA methyltransferase fibrillarin (Tollervey, Lehtonen et al., 1993). KD of Cent3 leads to accumulation of aberrant rRNA intermediates (Sanchez et al., 2016) and DEAD-box RNA helicases like Cent4 are often part of large complexes such as ribosomes or spliceosomes (Linder & Jankowsky, 2011). It is not known whether defects in ribosome biogenesis also affect SAT III transcript levels or vice versa, however the SAT III locus on the X chromosome is immediately adjacent to the rDNA locus and a deregulated chromatin state of one of the loci may directly or indirectly affect the other. Furthermore, there seems to be a strong connection between rRNA synthesis and GSC differentiation (Neumuller, Betschinger et al., 2008, Sanchez et al., 2016, Zhang, Shalaby et al., 2014. However, the fact that we did not observe any major defects in S2 cells and no obvious structural defects in the nucleolar organization argues against a major functional importance of the Centagon complex in ribosome biogenesis or nucleolar integrity. More work is needed to fully understand the precise relationship of centromeric transcript and the nucleolus, especially in specialized tissues like the germ line. KD of the Centagon members in early egg chambers resulted in egg chamber maturation defects, visible by an extended fragmented (5-blob) chromatin state in nurse cells, which remained small in size and eventually degenerated. A clear connection of the observed phenotypes with elevated SAT III RNA levels could be established: the simultaneous depletion of Centagon members and SAT III RNA reduced the nurse cell chromatin fragmentation phenotype and led to a higher number of eggs. Although the nurse cells do not undergo mitosis, they are subjected to a unique endocycle which requires a mitosis-like step to disperse the nurse cell chromatin after endocycle 5 (Dej & Spradling, 1999), which is apparently partially blocked by high SAT III RNA levels. Interestingly, some Centagon orthologues have been implicated in cell cycle functions: Cent1 orthologues in human and yeast and the human Cent2 orthologue promote S phase and replication initiation (Cheung, Amin et al., 2019, Johmura, Osada et al., 2008, Wang, Wang et al., 2020, Zhang, Yu et al., 2002 and the human orthologue of Cent3 promotes proliferation (Ma, Chang et al., 2008).
Germ cells are highly specialized cells in an organism that need to prepare the genome for transitioning from a fully differentiated germ cell to being part of the totipotent zygote after fertilization. This is reflected in massive chromatin remodelling events that must take place during early development to remove highly specialized chromatin organization from the sperm and egg to become a zygote. These early embryos are defined by a largely unconstrained genome conformations until the maternal to zygotic transition when the genome becomes ordered and structured into domains (Hug & Vaquerizas, 2018). We propose that an important function of the novel SAT III silencing mechanism we describe is to keep the transcriptional activation of repetitive elements under control during chromatin remodelling processes in the germ line to maintain genome integrity. This has been well described for the Drosophila germline, where transposable elements are transcriptionally silenced through heterochromatin formation mediated by the PIWI-interacting RNA pathway to protect the genome (Batki, Schnabl et al., 2019, Sienski, Donertas et al., 2012. We envision a similar scenario for repetitive regions that need to be highly controlled by a repressive complex in the germ line, in this case, Centagon. The complex is specifically expressed in the germ line and in tissues that undergo changes in their developmental potential, perhaps to contribute to satellite repeat silencing during chromatin remodelling phases that occur during these developmental stages. Why too much or too little SAT III RNA causes developmental defects is still unclear but by identifying the Centagon complex, we are one step closer in understanding SAT III RNA regulation and its specific role in germ line development.

RNA extraction
RNA from S2 cell pellets was purified using equivalent amount of Trizol (Invitrogen) or TriSure (Meridian Bioscience). Dissected and snap frozen ovaries were supplemented with Trizol (or TriSure) to a total of 100 μl. The tissue was homogenized and the samples were centrifuged at 12.000 x g at 4°C for 10 min. RNA was extracted from the supernatant according to the manufacturer's protocol. RNA pellets were resuspended in RNase-free water according to the pellet size (20-100 µl).
gDNA digestion of ovary RNA to eliminate DNA contamination. The reaction was mixed with 170 μl H2O as well as 20 μl NaAC (3M, pH 5.2) on ice, 220 μl Phenol-Chloroform-Isoamyl added, vortexed, and centrifuged at 16.000 x g for 5 min at RT. The upper phase was supplemented with 200 μl chloroform, vortexed and centrifuged at 16.000 x g 5 min at RT. The upper phase was mixed with 200 μl Isopropanol and 1 μl GlycoBlue (Invitrogen) at -80°C for at least 1 h. The sample was centrifuged and the pellet washed with 75% ethanol and again centrifuged, air-dry and the pellet resuspended in 15 μl H2O.
The RNA was heated at 65°C for 10 min to unfold, immediately placed on ice and loading and separated at 70 V in 1x MOPS (20 mM MOPS pH 7.0, 2 mM sodium acetate, 1 mM EDTA).

RNA Electromobility Shift Assay (EMSA)
Recombinant protein purification. GST-tagged protein in BL21 bacteria were grown to an OD 0.6 density and induced with 0.3 mM IPTG at 25°C for 16 h. Cells were harvested, washed with PBS, incubated in lysis buffer (500 mM NaCl, 0.1% (v/v) NP-40, 2mM PMSF, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin-Hemisulfate, 1 mg/ml Pepstatin, 1 mM DTT in 1x PBS) at 4°C and homogenized in two cycles with the Avestin Emulsiflex and centrifuged at 20.000 rpm at 4°C for 30 min. The lysate was filtered and loaded onto the ÄKTA GST column chromatography system, washes with 150 mM and 100 mM NaCl and eluted with 30 mM Glutathione in PBS, pH 9.0. The eluate was loaded onto a PD-10 desalting column (GE Healthcare) and eluted with PBS to remove salt. Further concentration was performed with a 10K centricon tube (Amnion).
Equal amounts of in vitro transcribed RNA (T7 MegaScript kit, Invitrogen) were incubated with increasing amounts of purified recombinant protein. RNA was heated to 68°C for 10 min and incubating 10 min at RT before placing on ice. The RNA and protein were cooled down for 30 min on ice and samples loaded on a non-denaturing 1% agarose gel in TAE buffer (40mM Tris, 20mM Acetate, 1mM EDTA) and run at 120 V followed by a 20 min ethidium bromide bath (RNase-free TAE).

Yeast Two Hybrid
All full length Centagon sequences were cloned into the pMM5-LexA DNA and pMM6-Gal4p TA vectors (Bade et al., 2014). A combination of one pMM5 and one pMM6 plasmid was transformed into competent yeastS SGY37VIII cells (Knop, Siegers et al., 1999). The YTH was performed as described previously (Bade et al., 2014). In short, interactions were judged based on the activity of â-galactosidase that results in the conversion of X-Gal (5-bromo-4chloro-3-indolyl-b-D-galactosidase) into a blue dye. Cell lysis and preclearing.
4xS1m-tagged RNA synthesis: For in vitro transcription, the Megascript kits for SP6 and T7 (Invitrogen) were used. In a 40 μl reaction, 2 μg of linearized plasmid (pSP73-4xS1m with different inserts) was used as a template and the reaction was left at 37°C overnight. The next day, 1 μl Turbo DNase was added and the reaction was incubated 15 more min at 37°C, followed by a clean-up with mini quick spin columns (Roche).

Immunofluorescent staining of cells
Immunofluorescence was performed as described (Mathew, Pauleau et al., 2014). Briefly, 3-5 x 10 5 cells were settled on Polysine Slides (Thermo Fisher), fixed with 4%, washed 3x 5 min with PBS and permeabilised with 0.1% Triton X-100 in PBS for 5 min. Cells were blocked with 4% BSA in PBS for 30 min followed by incubation with the primary antibody diluted in 4% BSA in PBS overnight. Cells were washed 3x with PBS before incubating with the fluorescent secondary antibody diluted 1:500 in 4% BSA in PBS for 1 h protected from light, washed 3x with PBS, counterstained with 1 µg/ml DAPI in PBS, washed again and mounted with Aqua-Poly/Mount (Polysciences). The following antibodies were used: mouse α-tubulin (1:1000, Sigma T9026) and rabbit α-Cenp-C (Pauleau, Bergner et al., 2019). Secondary fluorescent antibodies were purchased from Thermo Fisher Scientific. followed by secondary antibody for 2 h. The ovaries were rinsed once and washed twice for 15 min with PBST and counterstained with DAPI (1:000) in PBS. After two more 5 min PBST washes, the ovaries were mounted onto coverslips, with Aqua Polymount mounting medium.

Testis IF
Adult testes were dissected in PBS, fixed in 4% formaldehyde for 20 min and blocked in 5%    EMSA of SAT III sense and antisense RNA with BSA. In vitro transcribed RNA was incubated with BSA in the indicated molar ratios.
Expression data of the Centagon members per tissue according to www.flybase.org.
A. SAT III sense RNA FISH (green) in w 1118 , Zhr 1 and Mat67.15-Gal4-induced Cen3 KD ovaries, stained with DAPI (blue). SAT III RNA signals are depicted in one z-slice (middle) and as z-stack projection (right). The orange square enlarged on top shows the oocyte nucleus. For easier orientation, some images were rotated on top of a grey background.
Scale bar 50 μm. B. SAT III sense and antisense RNA FISH in w 1118 ovaries with or without RNase A digestion, stained with DAPI. The SAT III RNA FISH signal is shown as a z-stack projection. Scale bar 50 μm. C. SAT III antisense RNA FISH on newly hatched control (0-4 h old) and Centagon 1 KD (0-1 day old) ovaries. SAT III RNA signals are depicted in one zslice (middle) and as z-stack projection (right). Scale bar 50 μm.

Supplemental tables
Mass Spectromerty results of the SAT III RNA pulldowns Peptide counts in SAT III sense (S), antisense (AS) and 'loops only' (L) RNA pulldown.
log2(FC) = log2 values of the fold change of S/L, only proteins with a log2(FC) of 1 or higher are listed.