title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein.
abstract: Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5'-part is considered as an ESE binding SR proteins. Its 3'-part contains a decoy 5'-splice site (ss), which inhibits splicing at the bona fide 5'-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entire RSV NRS. Structural analysis of other avian NRSs and comparison with all sequenced avian NRSs is in favour of a phylogenetic conservation of the NRS 2D structure. By combination of approaches: (i) in vitro and in cellulo splicing assays, (ii) footprinting assays and (iii) purification and analysis of reconstituted RNP complex, we define a small NRS element retaining splicing inhibitory property. We also demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo. Altogether these data bring new insights on how NRS fine tune splicing activity.
authors.affiliation: ARN, RNP, Structure-Fonction-maturation, Enzymologie Moléculaire et Structurale, Faculté des Sciences et Techniques, Nancy Université-UMR 7214 CNRS-UHP, BP 70239, 54506 Vandoeuvre-lès-Nancy cedex, France.
authors.forename: Aileen
authors.initials: A
authors.lastname: Bar
authors.forename: Virginie
authors.initials: V
authors.lastname: Marchand
authors.forename: Georges
authors.initials: G
authors.lastname: Khoury
authors.forename: Natacha
authors.initials: N
authors.lastname: Dreumont
authors.forename: Annie
authors.initials: A
authors.lastname: Mougin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Robas
authors.forename: James
authors.initials: J
authors.lastname: Stévenin
authors.forename: Athanase
authors.initials: A
authors.lastname: Visvikis
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D052736:Rous sarcoma virus / Q000235:genetics*; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D000068103:Serine-Arginine Splicing Factors