Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

International audienceBackground: The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by b-arrestins, barr1 and barr2, which control both their signalling and endocytosis, suggesting that barrs may also function at primary cilium.Methodology/Principal Findings: In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.Conclusions/Significance: Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”

Trafic intracellulaire et ciliogénèse

Le cil primaire (CP) est présent dans presque la totalité de cellules des vertébrés, et des défauts de son assemblage/fonctionnement sont associés à des nombreuses ciliopathies. Le CP semble fonctionner comme une antenne mécanosensorielle dû à la présence de récepteurs dans la membrane ciliaire, comme les récepteurs couplés aux protéines G (RCPGs). Les beta-arrestines (barrs) 1 et 2 sont connues pour réguler les RCPGs membranaires, ce qui suggère qu'elles pourraient également réguler les RCPGs ciliaires. Nous avons observé que la parr2 est localisée au cil primaire de façon spécifique et qu'elle interagit avec Kif3A et 14-3-3, deux protéines importantes pour la ciliogenèse, suggérant un possible rôle directe sur la formation du cil. En effet, la parr2 joue un rôle sur la ciliogenèse, cependant, ceci semble être indirecte car son absence entraîne une surproliferation cellulaire, empêchant la formation du cil. Nous avons constaté que le cil est invaginé dans une poche ciliaire qui peut être transitoire ou permanente selon le type cellulaire d'où bourgeonnent de puits couverts de clathrine, ce qui nous a invité à étudier le rôle de complexes adaptateurs (AP) dans la ciliogenèse, observant que AP1 serait important pour la morphologie/orientation du cil. Ainsi, il est possible d'imaginer que cette poche ciliaire serve de plateforme d'arrimage de vésicules provenant du Golgi ainsi que pour de processus d'endocytose qui pourraient réguler des composant ciliaires.The primary cilium (PC) is present in almost all vertebrate cells and defects in its assembly/function are associated with a huge number of ciliopathies. PC seems to function as a mecanosensory antenna since is enriched in receptors, like the G-protein coupled receptors (RCPGs). Beta-arrestines (barrs) 1 and 2 regulate GPCR at the cell membrane, suggesting that they could also play a role in cilia-associated GPCRs. We found that barr2 is specifically localised to PC and that it interacts with Kif3A and 14-3-3, two proteins involved in ciliogenesis, suggesting a possible function of barr2 in ciliogenesis. Indeed, barr2 absence impedes PC formation. Nevertheless, this seems to be an indirect effect due to the fact that the absence of barr2 leads to a cell over proliferation, preventing cilia formation. We also observed that PC is invaginated in what we call the ciliary pocket, which can be transitory or permanent, depending on the cell type, from which clathrin coated pits bud forming clathrine coated vesicles. This led us to study the role of clathrin adaptor complexes (AP) in ciliogenesis, and we could observe that API would be important for the morphology/orientation of PC. Thus, it is possible to imagine that the ciliary pocket serves as a membrane platteform for the docking of Golgi-coming vesicles or for endocytic process which could control ciliary components.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Rhino breaks the deadlock in Drosophila testis

International audienceIn the early 2000s, Aravin and colleagues discovered, in the Drosophila melanogaster testis, a new class of small regulatory RNAs initially named repeat-associated small interfering RNA (rasiRNAs). rasiRNAs were first described as regulating a protein-coding gene. They are derived from the Suppressor of Stellate [Su(Ste)] locus located on the Y chromosome and were shown to target the X-linked Stellate repeated genes by sequence complementarity. Stellate genes encode proteins with homology to the regulatory β subunit of the protein kinase CK2. Stellate repression occurs during male gametogenesis and is essential for male fertility. These small RNAs were renamed as PIWI-interacting RNAs (pAU : Pleasenotethat}piRNAs}hasbeendefineda iRNAs); they are 23 to 29 nucleotides (nAU : Pleasenotethat}nt}hasbeendefinedas}nucleotides}inthesentence}ThesesmallRNAswererenamed t) long and bind to proteins of the PIWI family. Accordingly, piRNA pathway mutants are sterile, and they contain crystalline aggregates of Stellate-coded protein [1]. piRNAs were then discovered in mammals and in most other animal germ cells [2]. Nowadays, we know that piRNAs are mainly devoted to protecting the genome from active mobile genetic elements in the metazoan germline. In D. melanogaster, both sexes depend on a functional piRNA pathway for their fertility. Our understanding of piRNA biogenesis and function comes predominantly from studies of the female Drosophila germline, but the male's piRNA pathway remains poorly understood. Previously, studies reported that many proteins involved in the female piRNA pathway are also required for male fertility and Stellate silencing in the testis, supporting the conservation of piRNA pathway machinery in both sexes [3]. While Aub is expressed broadly from germline stem cells (GSCs) to primary spermatocytes, Ago3 and PIWI were detected only in mitotically dividing germline cells (GSCs and spermatogonia), indicating stage-specific modulations of the piRNA pathway [4-6] (Fig 1A). The 2 most active piRNA clusters in the testis are dual strand: Su(Ste) genes and AT-chX [5,7,8]. Interestingly, Aravin's team have recently defined novel piRNA clusters in D. melanogaster spermatogenesis and show piRNAs adaptation, dependent on sex-specific expression of transposons [8]. Dualstrand piRNA clusters generally lack promoters, and their expression depends on the Rhino-Deadlock-Cutoff (RDC) complex that licenses noncanonical transcription. The RDC is anchored to H3K9me3-marked chromatin via Rhino's chromodomain. This process involves 5 0-end protection of nascent RNAs and suppression of transcription termination [9]. Importantly, since fertility in male Rhino mutants was not compromised and stellate crystals were absent, RDC function in spermatogenesis was neglected [10]. In this issue of PLOS Genetics, Chen and colleagues explored whether piRNA loci in the D. melanogaster testis use the same RDC noncanonical transcription mechanism as in the female germline [11]. Unexpectedly, a detailed examination of male fertility by sperm exhaustion assays revealed germline defects and subfertility in RDC mutant males. A careful spatiotemporal analysis showed that the RDC complex is not ovary specific. RDC assembled also in early spermatogenesis to regulate piRNA cluster expression required for an efficient transposable element (TAU : Pleasenotethat}TE}hasbeendefinedas}transposableelement}inthesentence}RDCassem E) silencing in the testis. Like in ovaries, they observed that RDC marks all dua

tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription

International audienceRNase P is a conserved endonuclease that processes the 50 trailer of tRNA precursors. We have isolated mutations in Rpp30, a subunit of RNase P, and find that these induce complete sterility in Drosophila females. Here, we show that sterility is not due to a shortage of mature tRNAs, but that atrophied ovaries result from the activation of several DNA damage checkpoint proteins, including p53, Claspin, and Chk2. Indeed, we find that tRNA processing defects lead to increased replication stress and de-repression of transposable elements in mutant ovaries. We also report that transcription of major piRNA sources collapse in mutant germ cells and that this correlates with a decrease in heterochromatic H3K9me3 marks on the corresponding piRNA-producing loci. Our data thus link tRNA processing, DNA replication, and genome defense by small RNAs. This unexpected connection reveals constraints that could shape genome organization during evolution

tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation

International audiencetRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. Here we describe a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using our pipeline, we show how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology