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| BELLE localizes in the main pacemaker cells in the brain of Drosophila melanogaster. Confocal images of whole mount of adult brain expressing BELLE fused with green fluorescent protein (bel::GFP). Scale bar: 10 µm. (A,D) Anti-GFP immunostaining (BEL::GFP). (B,E) Anti-PDF was used to label PDF-expressing ventral lateral neurons. Depends on optical section small LNv (s-LNv) or large LNv (l-LNv) are visualized. (C,F) BEL::GFP and PDF immune staining co-localize in the area of LNv cell bodies.
Source publication
Circadian clocks control and synchronize biological rhythms of several behavioral and physiological phenomena in most, if not all, organisms. Rhythm generation relies on molecular auto-regulatory oscillations of interlocked transcriptional-translational feedback loops. Rhythmic clock-gene expression is at the base of rhythmic protein accumulation,...
Contexts in source publication
Context 1
... were maintained in standard light-dark conditions, brains were dissected and an anti-GFP antibody was used to detect BEL::GFP expression. We observed BELLE expression in clock neurons and in glial cells in the optic lobe, with a predominantly cytoplasmic localization (Figures 2, 3). In clock neurons, BELLE was expressed in either the small or the large ventral lateral neurons (s-LNvs and l-LNvs respectively) (Figure 2), PDF-expressing cells that act in promoting and governing the circadian locomotor behavior. ...
Context 2
... observed BELLE expression in clock neurons and in glial cells in the optic lobe, with a predominantly cytoplasmic localization (Figures 2, 3). In clock neurons, BELLE was expressed in either the small or the large ventral lateral neurons (s-LNvs and l-LNvs respectively) (Figure 2), PDF-expressing cells that act in promoting and governing the circadian locomotor behavior. In glia we observed BELLE expression in the epithelial glia and a weak labeling also in the lamina (Figures 3D-F): position and shape of these GFP-labeled cells suggest they may be R1-R6 photoreceptor terminals ( Figures 3D-F); however, anti-REPO staining showed glia cells nuclei at the top of each structure ( Figure 3G). ...
Context 3
... Anti-GFP staining on section of Rh1 > GFP brains showing R1-R6 photoreceptors terminals in the lamina. Figure S2). This result is in line with that obtained by co-immunoprecipitation ( Figure 1B): the lower amount of BELLE pulled down in light compared to dark uniquely depends on the light-mediated degradation of CRY. ...
Citations
... In this way, the expression patterns of the clock components induce oscillatory behaviours in their downstream interactants [94,124]. It was also shown that alternative splicing, as well as piRNA-mediated regulation of the transposons, could represent another level of clock control [129]. The CC, in general, is susceptible to stressthe circadian cortisol-mediated entrainment of ultradian transcription pulses that provide the normal feedback regulation of cellular function is then lost [130]. ...
Here, we review the role of the circadian clock (CC) in the resistance of cancer cells to genotoxic treatments in relation to whole-genome duplication (WGD) and telomere-length regulation. The CC drives the normal cell cycle, tissue differentiation, and reciprocally regulates telomere elongation. However, it is deregulated in embryonic stem cells (ESCs), the early embryo, and cancer. Here, we review the DNA damage response of cancer cells and a similar impact on the cell cycle to that found in ESCs—overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, coupling telomere erosion to accelerated cell senescence, and favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Polyploidy decelerates the CC. We report an intriguing positive correlation between cancer WGD and the deregulation of the CC assessed by bioinformatics on 11 primary cancer datasets (rho = 0.83; p < 0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by ALT-recombination, and return their depolyploidised offspring to telomerase-dependent regulation. By reversing this polyploidy and the CC “death loop”, the mitotic cycle and Hayflick limit count are thus again renewed. Our review and proposed mechanism support a life-cycle concept of cancer and highlight the perspective of cancer treatment by differentiation.
... In this way, the expression patterns of the clock components induce oscillatory behaviours in their downstream interactants [94,124]. It was also shown that alternative splicing, as well as piRNA-mediated regulation of the transposons, could represent another level of clock control [129]. The CC, in general, is susceptible to stressthe circadian cortisol-mediated entrainment of ultradian transcription pulses that provide the normal feedback regulation of cellular function is then lost [130]. ...
p>Here, we review the role of the circadian clock (CC) in the resistance of cancer cells to genotoxic treatments in relation to whole-genome duplication (WGD) and telomere-length regulation. The CC drives the normal cell cycle, tissue differentiation, and reciprocally regulates telomere elongation. However, it is deregulated in embryonic stem cells (ESCs), the early embryo, and cancer. Here, we review the DNA damage response of cancer cells and a similar impact on the cell cycle to that found in ESCs—overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, coupling telomere erosion to accelerated cell senescence, and favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Polyploidy decelerates the CC. We report an intriguing positive correlation between cancer WGD and the deregulation of the CC assessed by bioinformatics on 11 primary cancer datasets (rho = 0.83; p < 0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by ALT-recombination, and return their depolyploidised offspring to telomerase-dependent regulation. By reversing this polyploidy and the CC “death loop”, the mitotic cycle and Hayflick limit count are thus again renewed. Our review and proposed mechanism support a life-cycle concept of cancer and highlight the perspective of cancer treatment by differentiation.</p
... ; https://doi.org/10.1101/2022.01.14.476363 doi: bioRxiv preprint expression patterns of the clock components induce oscillatory behaviours in their downstream interactants [57,78]. It was also shown that alternative splicing, as well as piRNA-mediated regulation of the transposons could represent another level of clock control [83]. The CC in general is susceptible to stress conditions -the circadian cortisolmediated entrainment of ultradian transcription pulses that provide the normal feedback regulation of cellular function is then lost [84]. ...
The Circadian Clock (CC) drives the normal cell cycle and reciprocally regulates telomere elongation. However, it can be deregulated in cancer, embryonic stem cells (ESC) and the early embryo. Here, its role in the resistance of cancer cells to genotoxic treatments was assessed in relation to whole-genome duplication (WGD) and telomere regulation. We first evaluated the DNA damage response of polyploid cancer cells and observed a similar impact on the cell cycle to that seen in ESC - overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, and coupling telomere erosion to accelerated cell senescence, favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Next, we revealed a positive correlation between cancer WGD and deregulation of CC assessed by bioinformatics on 11 primary cancer datasets (rho=0.83; p<0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by recombination and return their depolyploidised mitotic offspring to TERT-dependent telomere regulation. Through depolyploidisation and the CC "death loop", the telomeres and Hayflick limit count are thus again renewed. This mechanism along with similar inactivity of the CC in early embryos supports a life-cycle (embryonic) concept of cancer.
... The Drosophila orthologue, armitage (armi; Cook et al., 2004) is involved in the RNAi pathway, required for genome stability in the ovary, where silencing is needed to establish the oocyte cytoskeleton polarity during early oogenesis. The main role of Armi is in the biogenesis of primary piRNAs in the somatic cells of the gonads (Lim and Kai, 2007;Olivieri et al., 2010), where it interacts with several components of the piRNA pathway, thus being crucial for the genome stability and fertility of both sexes (Megosh et al., 2006;Saito et al., 2010;Bozzetti et al., 2015;Specchia et al., 2017;Cusumano et al., 2019;Durdevic and Ephrussi, 2019;Ge et al., 2019a;Ishizu et al., 2019;Specchia et al., 2019). Indeed, armitage mutants support the early stages in the RNAi pathway but fail in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi (Tomari et al., 2004). ...
STUDY QUESTION
Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function?
SUMMARY ANSWER
This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis.
WHAT IS KNOWN ALREADY
POI, a condition occurring in 1% of women under 40 years of age, affects women’s fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated.
STUDY DESIGN, SIZE, DURATION
WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The Italian POI patients’ DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility.
MAIN RESULTS AND THE ROLE OF CHANCE
The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility.
LARGE SCALE DATA
The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375.
LIMITATIONS, REASONS FOR CAUTION
This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents’ DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients’ phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis.
WIDER IMPLICATIONS OF THE FINDINGS
The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways).
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by Italian Ministry of Health grants ‘Ricerca Corrente’ (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by ‘Piano Sostegno alla Ricerca’ (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.
... Besides this role as a circadian repressor, an involvement of CRY in the posttranscriptional control of the circadian clock can also be hypothesized. Indeed, we have recently shown that CRY interacts with BELLE (Cusumano et al., 2019), a DEAD-box RNA helicase essential for viability and fertility (Johnstone et al., 2005), and plays important functions in RNA metabolism, from splicing and translational regulation to miRNA and siRNA pathways (Worringer et al., 2009;Pek and Kai, 2011;Ihry et al., 2012). We have observed an involvement of BELLE in circadian rhythmicity and in the piRNA-mediated regulation of transposable elements, suggesting that this specific posttranscriptional mechanism could be in place to ensure proper rhythmicity (Cusumano et al., 2019). ...
... Indeed, we have recently shown that CRY interacts with BELLE (Cusumano et al., 2019), a DEAD-box RNA helicase essential for viability and fertility (Johnstone et al., 2005), and plays important functions in RNA metabolism, from splicing and translational regulation to miRNA and siRNA pathways (Worringer et al., 2009;Pek and Kai, 2011;Ihry et al., 2012). We have observed an involvement of BELLE in circadian rhythmicity and in the piRNA-mediated regulation of transposable elements, suggesting that this specific posttranscriptional mechanism could be in place to ensure proper rhythmicity (Cusumano et al., 2019). ...
Cryptochromes (CRYs) are flavoproteins that are sensitive to blue light, first identified in Arabidopsis and then in Drosophila and mice. They are evolutionarily conserved and play fundamental roles in the circadian clock of living organisms, enabling them to adapt to the daily 24-h cycles. The role of CRYs in circadian clocks differs among different species: in plants, they have a blue light-sensing activity whereas in mammals they act as light-independent transcriptional repressors within the circadian clock. These two different functions are accomplished by two principal types of CRYs, the light-sensitive plant/insect type 1 CRY and the mammalian type 2 CRY acting as a negative autoregulator in the molecular circadian clockwork. Drosophila melanogaster possesses just one CRY, belonging to type 1 CRYs. Nevertheless, this single CRY appears to have different functions, specific to different organs, tissues, and even subset of cells in which it is expressed. In this review, we will dissect the multiple roles of this single CRY in Drosophila, focusing on the regulatory mechanisms that make its pleiotropy possible.
... It has been shown that bel is essential for fly viability [17]. Belle protein is expressed ubiquitously and possesses extremely high functional pleiotropy in fly tissues, being involved in the ecdysone-triggered transcriptional cascade during metamorphosis [18]; in the small RNA-based silencing mechanisms [19,20]; in the Notchinduced differentiation of ovarian follicle cells and establishment of correct anterior-posterior polarity of the developing oocyte [21]; in the proper chromosome segregation during mitotic anaphase in S2 cells [22]; in the circadian rhythmicity in Drosophila neurons [23]; and in the cell cycle regulation in somatic tissues and the germline [24][25][26]. RNA helicase Belle is shown to regulate the translation of a set of specific mRNAs in an ATP-dependent manner [18,[26][27][28]. ...
DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Belle is involved both in translational activation and repression of target mRNAs in different tissues; however, direct targets of Belle in the testes are essentially unknown. Here we showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of β-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail we performed cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis and identified multiple mRNAs that interacted with Belle in the testes. The set of Belle targets includes transcripts of proteins that are essential for preventing the tumor-like clusters of germ cells and for sustaining spermatogenesis. By our hypothesis, failures in the translation of a number of mRNA targets additively contribute to developmental defects observed in the testes with belle knockdowns both in cyst cells and in the germline.
