Nxt1 Is Necessary for the Terminal Step of Crm1-Mediated Nuclear Export

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor–substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616–8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm

Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow

The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre-established mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whitlock/Witte-type culture) or in the presence of 10(-7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cells accounting for 50% to 90% of the organ-cultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3-/low and TCRalphabeta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus

Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

Export nucléaire des protéines et homéostasie cellulaire

L’apparition du noyau dans les cellules eucaryotes a imposé la mise en place de mécanismes spécifiques permettant d’utiliser et de protéger l’information génétique mais également de coordonner les fonctions nucléaires et cytoplasmiques. Les études menées au cours des 10 dernières années ont permis d’élaborer un modèle selon lequel les molécules importées vers le noyau ou exportées vers le cytoplasme présentent des séquences d’adressage reconnues par des récepteurs spécifiques de transport nucléaire. Ces interactions sont orchestrées par la petite GTPase Ran qui assure la directionnalité des échanges nucléocytoplasmiques. Cet article tentera de faire le point des connaissances sur les mécanismes d’export nucléaire des protéines et le rôle de ces voies de transport dans la régulation d’autres fonctions cellulaires.The nucleus of eukaryotic cells spatially separates DNA replication and RNA transcription from cytoplasmic protein synthesis. Thus, the nuclear membrane must ensure a strict and selective molecular control of exchanges between the nucleus and the rest of the cell, not only to protect and correctly transmit genetic information but also to synchronize nuclear and cytoplasmic functions. Studies over the past ten years led to the identification of targeting sequences (nuclear import or export sequences) recognized by specific receptors, called importins and exportins, which induce transport through the nuclear pore complex. Cargo-receptor interactions are orchestrated by Ran, a small and abundant GTPase. The compartmentalization of the factors that control the GDP- and GTP-bound state of Ran is believed to create a steep gradient of RanGDP (cytoplasmic) / RanGTP (nuclear) concentrations across the nuclear membrane. This gradient controls the directionality of nucleocytoplasmic transport pathways since cargo-importin complexes are induced to disassemble upon binding to RanGTP in the nucleus whereas RanGTP is used to assemble cargo-exportin complexes. In this review, we focus on what is known about the various steps involved in nuclear export of proteins, an intracellular route discovered more than 40 years ago, which has remained largely uncharacterized until recently. Furthermore, the regulation of nuclear transport is now considered one of the most efficient mechanisms to adapt cellular responses to environment conditions by restricting access to nuclear or cytoplasmic compartments. In particular, we illustrate here how nuclear export regulation participates to the control of transcriptional responses, cell cycle progression or the establishment of cell polarity

Propriétés stucturales et fonctionnelles du domaine UBA de Mex67 (le récepteur d'export nucléaire des ARNm)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Contrôle de la localisation intracellulaire et du métabolisme de deux protéines des complexes de pré-intégration du VIH-1, l'intégrase et Vpr

PARIS-BIUSJ-Thèses (751052125) / SudocCACHAN-ENS (940162301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways.

International audienceThe AAA-ATPase Cdc48/p97 controls a large array of cellular functions including protein degradation, cell division, membrane fusion through its ability to interact with and control the fate of ubiquitylated proteins. More recently, Cdc48/p97 also appeared to be involved in autophagy, a catabolic cell response that has long been viewed as completely distinct from the Ubiquitine/Proteasome System. In particular, conjugation by ubiquitin or ubiquitin-like proteins as well as ubiquitin binding proteins such as Cdc48/p97 and its cofactors can target degradation by both catabolic pathways. This review will focus on the recently described functions of Cdc48/p97 in autophagosome biogenesis as well as selective autophagy. This article is part of a Special Issue entitled: AAA ATPases: structure and function