Structure of the Pre-mRNA Leakage 39-kDa Protein Reveals a Single Domain of Integrated zf-C3HC and Rsm1 Modules

In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix αC in the zf-C3HC and Rsm1 modules, and an extra helix αAB\u27 in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae

The Ultimate (Mis)match: When DNA Meets RNA

International audienceThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC B

Propriétés de l'ARN polymérase II et de sa phosphatase FCP1 dans l'embryon précoce de Xénope

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

Etude de la régulation du métabolisme des ARN messagers chez la levure Saccharomyces cerevisiae

Au cours de la transcription, plusieurs facteurs sont assemblés sur les ARN messagers pour former des Ribonucléoparticules de messagers (mRNPs), et contrôler leur maturation, leur stabilité et leur devenir dans le cytoplasme. Afin d assurer la production de protéines fonctionnelles, la cellule dispose de plusieurs mécanismes de régulation et de contrôle de qualité assurant la fidélité de l information génétique transmise au niveau ARN messager et protéine.Chez la levure Saccharomyces cerevisiae, un ensemble de protéines associées au pore nucléaire, incluant la SUMO protéase Ulp1, a été impliqué dans un contrôle de qualité des mRNPs régulant leur export vers le cytoplasme. Ces données suggéraient que l export des ARN messagers pourrait être contrôlé par la modification post-traductionnelle par le polypeptide SUMO d un ou de plusieurs effecteurs au sein des mRNPs. Afin de mieux comprendre ces processus, nous avons combiné plusieurs approches visant à identifier ces protéines SUMOylées. En particulier, nous avons mis en place un crible protéomique visant à identifier les protéines dont l association sur les mRNPs dépend d Ulp1. Ce crible nous a permis de mettre en évidence une régulation par Ulp1 de l assemblage du complexe THO sur les ARN messagers. Ce complexe, recruté sur les gènes et les mRNPs, est connu pour contribuer à l efficacité de la transcription, prévenir l instabilité génétique liée à la formation d hybrides ADN matrice ARN messager (dénommés R-loops) et permettre l export des mRNPs. En combinant l analyse biochimique de différentes catégories de mRNPs à des expériences d immunoprécipitation de l ARN, nous avons montré que l activité de la SUMO-protéase Ulp1 est nécessaire à l association du complexe THO sur différents ARN messagers. De plus, nous avons montré que le complexe THO est SUMOylé sur le domaine C-terminal de sa sous-unité Hpr1, et que Ulp1 régule cette modification. Enfin, cet événement de SUMOylation du complexe THO régule son association avec les mRNPs. L analyse fonctionnelle de mutants affectant la SUMOylation du complexe THO révèle que des défauts de SUMOylation de ce complexe compromettent ses fonctions dans la transcription sans affecter l export. De manière intéressante, nous avons observé que la présence d un intron sur des rapporteurs LacZ diminue la sensibilité de leur expression à des inactivations ou des défauts de SUMOylation du complexe THO. Ce phénotype entraine une augmentation relative des niveaux d ARN pré-messagers dans ces mutants, un phénomène rendant compte de la fuite cytoplasmique apparente d ARN non épissés précédemment observée dans le mutant ulp1. L ensemble de ces données caractérise pour la première fois un rôle de la SUMOylation dans le contrôle de l assemblage et du devenir cellulaire des mRNPs.During transcription, several factors associate with mRNA to form messenger Ribonucleoparticles (mRNPs), thereby controlling their processing, their stability, and their cytoplasmic fate. To ensure the production of functional proteins from these mRNAs, eukaryotic cells contain numerous regulatory and quality control systems in order to prevent aberrant mRNP accumulation and export.In the yeast Saccharomyces cerevisiae, several nuclear pore associated proteins, including the SUMO isopeptidase Ulp1, have been involved in a mRNP quality control regulating their nuclear export. These data suggested that post-translational modification by SUMO of one or several mRNP components could regulate mRNA export. In order to understand the molecular mechanisms underlying this process, we undertook several approaches to identify these SUMOylated factors. In particular, we have set up a proteomic screen to identify mRNP components whose assembly onto mRNPs depends on Ulp1 activity.This proteomic survey revealed an Ulp1-dependent regulation of THO complex assembly to mRNPs. This complex, recruited to transcribed genes and mRNPs, is known to regulate transcription elongation by preventing DNA-RNA hybrids formation (termed R-loops), and mRNP export. Through a combination of proteomic analysis of mRNPs assembled in Ulp1 mutant cells, with RNA / chromatin immunoprecipitation experiments, we demonstrate that Ulp1 controls specifically the recruitment of the THO complex within mRNPs. SUMOylation analysis further reveals that Ulp1 targets the THO complex subunit Hpr1 on its C-terminal domain for deSUMOylation. We further show that this SUMOylation event regulates THO complex association within mRNPs. Finally, functional analysis reveal that impaired deSUMOylation of the THO complex do not affect mRNP export, but disturbs expression of LacZ reporter genes, a phenotype classically associated with THO complex dysfunction. Intriguingly, the transcriptional effect of inactivation or impaired deSUMOylation of the THO complex on LacZ expression is alleviated by the presence of an intron, providing a molecular basis for previously reported pre-mRNA leakage phenotypes. Our data therefore unravels for the first time a function of SUMO in the control of mRNP assembly contributing to proper mRNP homeostasis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Pml39, a Novel Protein of the Nuclear Periphery Required for Nuclear Retention of Improper Messenger Ribonucleoparticles

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60Δ mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39Δ partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal

International audienceNuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences

A single aspartate mutation in the conserved catalytic site of Rev3L generates a hypomorphic phenotype in vivo and in vitro

International audienceRev3, the catalytic subunit of yeast DNA polymerase ζ, is required for UV resistance and UV-induced mutagenesis, while its mammalian ortholog, REV3L, plays further vital roles in cell proliferation and embryonic development. To assess the contribution of REV3L catalytic activity to its in vivo function, we generated mutant mouse strains in which one or two Ala residues were substituted to the Asp of the invariant catalytic YGDTDS motif. The simultaneous mutation of both Asp (ATA) phenocopies the Rev3l knockout, which proves that the catalytic activity is mandatory for the vital functions of Rev3L, as reported recently. Surprisingly, although the mutation of the first Asp severely impairs the enzymatic activity of other B-family DNA polymerases, the corresponding mutation of Rev3 (ATD) is hypomorphic in yeast and mouse, as it does not affect viability and proliferation and moderately impacts UVC-induced cell death and mutagenesis. Interestingly, Rev3l hypomorphic mutant mice display a distinct, albeit modest, alteration of the immunoglobulin gene mutation spectrum at G-C base pairs, further documenting its role in this process