title: Differential effects of the SR proteins 9G8, SC35, ASF/SF2, and SRp40 on the utilization of the A1 to A5 splicing sites of HIV-1 RNA.
abstract: Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.
authors.affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR CNRS 7567, Université Henri Poincaré Nancy 1, Boulevard des Aiguillettes, BP239, 54506 Vandoeuvre-lès-Nancy, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Ropers
authors.forename: Lilia
authors.initials: L
authors.lastname: Ayadi
authors.forename: Renata
authors.initials: R
authors.lastname: Gattoni
authors.forename: Sandrine
authors.initials: S
authors.lastname: Jacquenet
authors.forename: Laurence
authors.initials: L
authors.lastname: Damier
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
authors.forename: James
authors.initials: J
authors.lastname: Stévenin
mesh_terms: D017398:Alternative Splicing; D001665:Binding Sites; D015153:Blotting, Western; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D012367:RNA, Viral; D016601:RNA-Binding Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic
chemical_list: D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C405483:U1 small nuclear RNA; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors