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

tRNA 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.

Monitoring ß-Arrestin 2 Targeting to the Centrosome, Basal Body, and Primary Cilium by Fluorescence Microscopy.

Primary cilia (PC) are microtubule-based organelles that behave like a cellular antenna controlling key signaling pathways during development and tissue homeostasis. The ciliary membrane is highly enriched for G protein-coupled receptors (GPCRs), and PC are a crucial signaling compartment for this large receptor family. Downstream effectors of GPCR signaling are also present in cilia, and evidence obtained by our labs and others demonstrated that ß-arrestin (ßarr) family members are differentially recruited to PC and have investigated the role of GPCR activation in this process. In this chapter, we provide methods based on fluorescence microscopy on fixed or live cells suitable for investigating targeting and recruitment of ßarrs at PC.

The replicative histone chaperone CAF1 is essential for the maintenance of identity and genome integrity in adult stem cells.

Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we have investigated the role of CAF1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF1 play an important role in both the regulation of stem cell identity and genome integrity.

Morphological and Functional Characterization of the Ciliary Pocket by Electron and Fluorescence Microscopy.

In many vertebrate cell types, the proximal part of the primary cilium is positioned within an invagination of the plasma membrane known as the ciliary pocket. Recent evidence points to the conclusion that the ciliary pocket comprises a unique site for exocytosis and endocytosis of ciliary proteins, which regulates the spatiotemporal trafficking of receptors into and out of the cilium to control its sensory function. In this chapter, we provide methods based on electron microscopy, 3D reconstruction of fluorescence images as well as live cell imaging suitable for investigating processes associated with endocytosis at the ciliary pocket.

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

RNase P is a conserved endonuclease that processes the 5' 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.

Chromatin modifications regulate germ cell development and transgenerational information relay.

Germ cells transmit genetic, cytoplasmic and epigenetic information to the next generation. Recent reports describe the importance of chromatin modifiers and small RNAs for germ cells development in Drosophila. We also review exciting progress in our understanding of piRNAs functions, which demonstrate that this class of small RNAs is both an adaptive and inheritable epigenetic memory.

A mosaic genetic screen for genes involved in the early steps of Drosophila oogenesis.

The first hours of Drosophila embryogenesis rely exclusively on maternal information stored within the egg during oogenesis. The formation of the egg chamber is thus a crucial step for the development of the future adult. It has emerged that many key developmental decisions are made during the very first stages of oogenesis. We performed a clonal genetic screen on the left arm of chromosome 2 for mutations affecting early oogenesis. During the first round of screening, we scored for defects in egg chambers morphology as an easy read-out of early abnormalities. In a second round of screening, we analyzed the localization of centrosomes and Orb protein within the oocyte, the position of the oocyte within the egg chamber, and the progression through meiosis. We have generated a collection of 71 EMS-induced mutants that affect oocyte determination, polarization, or localization. We also recovered mutants affecting the number of germline cyst divisions or the differentiation of follicle cells. Here, we describe the analysis of nine complementation groups and eight single alleles. We mapped several mutations and identified alleles of Bicaudal-D, lethal(2) giant larvae, kuzbanian, GDP-mannose 4,6-dehydratase, tho2, and eiF4A. We further report the molecular identification of two alleles of the Drosophila homolog of Che-1/AATF and demonstrate its antiapoptotic activity in vivo. This collection of mutants will be useful to investigate further the early steps of Drosophila oogenesis at a genetic level.

The ciliary pocket: a once-forgotten membrane domain at the base of cilia.

The PC (primary cilium) is present on most cell types in both developing and adult tissues in vertebrates. Despite multiple reports in the 1960s, the PC was almost forgotten for decades by most of the cell biology community, mainly because its function appeared enigmatic. This situation changed 10 years ago with the key discovery that this fascinating structure is the missing link between complex genetic diseases and key signalling pathways during development and tissue homoeostasis. A similar misfortune might have happened to an original membrane domain found at the base of PC in most cell types and recently termed the 'ciliary pocket'. A morphologically related structure has also been described at the connecting cilium of photoreceptors and at the flagellum in spermatids. Its organization is also reminiscent of the flagellar pocket, a plasma membrane invagination specialized in uptake and secretion encountered in kinetoplastid protozoa. The exact function of the ciliary pocket remains to be established, but the recent observation of endocytic activity coupled to the fact that vesicular trafficking plays important roles during ciliogenesis brought excitement in the ciliary community. Here, we have tried to decipher what this highly conserved membrane domain could tell us about the function and/or biogenesis of the associated cilium.

The AP-1 clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport.

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control.

BACKGROUND: 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 beta-arrestins, betaarr1 and betaarr2, which control both their signalling and endocytosis, suggesting that betaarrs may also function at primary cilium. METHODOLOGY/PRINCIPAL FINDINGS: In cycling cells, betaarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, betaarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, betaarr2 was found at the basal body and axoneme of primary cilia. Interestingly, betaarr2 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, betaarrs appear to control cell cycle progression. Indeed, cells lacking betaarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions. CONCLUSIONS/SIGNIFICANCE: Our results show that betaarr2 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, betaarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell "antenna".

Clinical, cellular, and neuropathological consequences of AP1S2 mutations: further delineation of a recognizable X-linked mental retardation syndrome.

Mutations in the AP1S2 gene, encoding the sigma1B subunit of the clathrin-associated adaptor protein complex (AP)-1, have been recently identified in five X-linked mental retardation (XLMR) families, including the original family with Fried syndrome. Studying four patients in two unrelated families in which AP1S2 nonsense and splice-site mutations segregated, we found that affected individuals presented, in addition to previously described features, with elevated protein levels in cerebrospinal fluid (CSF). Moreover, computed tomography scans demonstrated that the basal ganglia calcifications associated with AP1S2 mutations appeared during childhood and might be progressive. Based on these observations, we propose that AP1S2 mutations are responsible for a clinically recognizable XLMR and autism syndrome associating hypotonia, delayed walking, speech delay, aggressive behavior, brain calcifications, and elevated CSF protein levels. Using the AP-2 complex, in which the sigma subunit is encoded by one single gene, as a model system, we demonstrated that sigma subunits are essential for the stability of human AP complexes. By contrast, no major alteration of the stability, subcellular localization, and function of the AP-1 complex was observed in fibroblasts derived from a patient carrying an AP1S2 mutation. Similarly, neither macro- nor microscopic defects were observed in the brain of an affected fetus. Altogether, these data suggest that the absence of an AP-1 defect in peripheral tissues is due to functional redundancy among AP-1 sigma subunits (sigma1A, sigma1B, and sigma1C) and that the phenotype observed in our patients results from a subtle and brain-specific defect of the AP-1-dependent intracellular protein traffic.

Intracellular trafficking of CD23: differential regulation in humans and mice by both extracellular and intracellular exons.

In mouse models of food allergy, we recently characterized a new CD23b-derived splice form lacking extracellular exon 5, bDelta5, which undergoes constitutive internalization and mediates the transepithelial transport of free IgE, whereas classical CD23b is more efficient in transporting IgE/allergen complexes. These data suggested that regulation of endocytosis plays a central role in CD23 functions and drove us to systematically compare the intracellular trafficking properties of human and murine CD23 splice forms. We found that CD23 species show similar endocytic behaviors in both species; CD23a undergoes constitutive clathrin-dependent internalization, whereas CD23b is stable at the plasma membrane. However, the mechanisms controlling these similar behaviors appeared to be different. In mice, a positive internalization signal was localized in the cytoplasmic region shared by all CD23 splice forms. This positive signal was negatively regulated by the intracellular CD23b-specific exon. In addition, the fact that alternative splice forms lacking exons of the extracellular region (5, 6, 7, and/or 8) were all constitutively internalized suggested that endocytosis of murine CD23 is regulated by a process similar to the outside-in signaling of integrins. In humans, the internalization signal was mapped in the CD23a-specific intracellular exon. Interestingly, this signal also behaved as a basolateral targeting signal in polarized Madin-Darby canine kidney cells. The latter result and the fact that human intestinal cell lines were found to coexpress both CD23a and CD23b provide a molecular explanation for the initial observations that CD23 was found at the basolateral membrane of intestinal epithelial cells from allergic patients.