The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element.
The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3' splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.
Tange
T O
TO
Department of Molecular and Structural Biology, University of Aarhus, C.F. Møllers Allé, Building 130, DK-8000 Arhus C, Denmark.
Damgaard
C K
CK
Guth
S
S
Valcárcel
J
J
Kjems
J
J
D017398:Alternative Splicing / Q000502:physiology*; D001483:Base Sequence; D002474:Cell-Free System; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D015696:Gene Products, tat / Q000502:physiology*; D020868:Gene Silencing; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011485:Protein Binding; D021122:Protein Subunits; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism / Q000502:physiology*; D017381:Spliceosomes / Q000378:metabolism; D000072668:Splicing Factor U2AF; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021122:Protein Subunits; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D054322:tat Gene Products, Human Immunodeficiency Virus