title: Tra2-mediated recognition of HIV-1 5' splice site D3 as a key factor in the processing of vpr mRNA.
abstract: Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3' splice site (3'ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5'ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE(vpr)) localized between exonic splicing silencer ESSV and 5'ss D3. The ESE(vpr) sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE(vpr). Further analyses revealed that ESE(vpr) supports the binding of U1 snRNA at 5'ss D3, allowing bridging interactions across the upstream exon with 3'ss A2. In line with this, an increase or decrease in the complementarity of 5'ss D3 to the 5' end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3'ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5'ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3'ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.
authors.affiliation: Institute for Virology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Erkelenz
authors.forename: Gereon
authors.initials: G
authors.lastname: Poschmann
authors.forename: Stephan
authors.initials: S
authors.lastname: Theiss
authors.forename: Anja
authors.initials: A
authors.lastname: Stefanski
authors.forename: Frank
authors.initials: F
authors.lastname: Hillebrand
authors.forename: Marianne
authors.initials: M
authors.lastname: Otte
authors.forename: Kai
authors.initials: K
authors.lastname: Stühler
authors.forename: Heiner
authors.initials: H
authors.lastname: Schaal
mesh_terms: D017398:Alternative Splicing; D002460:Cell Line; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051916:Membrane Cofactor Protein / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D022821:RNA Splice Sites / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: C497857:CD46 protein, human; D051916:Membrane Cofactor Protein; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012367:RNA, Viral; C405483:U1 small nuclear RNA; D054325:vpr Gene Products, Human Immunodeficiency Virus