RNA splicing at human immunodeficiency virus type 1 3' splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element - PubMed (original) (raw)

RNA splicing at human immunodeficiency virus type 1 3' splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element

P S Bilodeau et al. J Virol. 2001 Sep.

Abstract

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

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Figures

FIG. 1

FIG. 1

(A) Structure of the HIV-1 NL4-3 genome. Boxes indicate open reading frames. Hash marks represent endpoints of gag-pol deletion in pΔPSP. ESS sequences are shown by shaded boxes. Oligonucleotide primers used are indicated by arrows designating position and orientation. LTR, long terminal repeat. (B) Structures of the small (∼1.8-kb) and intermediate (∼4.0-kb) size classes of HIV-1 transcripts. Exons are indicated as black bars. The exons within the different RNA species are designated by numbers (and sometimes letters) within the boxes according to the nomenclature of Purcell and Martin (35). The exon designations with the letter I indicate exons present only in the intermediate-size HIV-1 mRNA species. The alternative noncoding exons 2 and 3 are indicated with asterisks. Locations of 5′ (D) and 3′ (A) splice sites are shown.

FIG. 2

FIG. 2

Mutagenesis of 5′ splice site D3 toward the consensus 5′ splice site sequence (panels A and B) and putative silencers (panels C and D) increases splicing at 3′ splice site A2 and inclusion of noncoding exon 2. RT-PCR analyses of ∼4.0-kb (A and C) and ∼1.8-kb (B and D) mRNAs from cells transfected with wild-type and mutant plasmids (pΔPSP and pSPD3up in panels A and B; pΔPSP and pSPRS in panels C and D) were performed. Mock-transfected cell mRNA was analyzed in parallel. Denaturing PAGE of 32P-labeled PCR products was performed as described in Materials and Methods. The mutated sequences in each case are shown below the wild-type NL4-3 sequence, and the changes are underlined. The PyUAG sequences are shown in italic type. RNA species are designated with exon numbers (and letters) as in Fig. 1. Note in panels A and C that RNA species 1.3.4aI and 1.3.4bI were not separated. The locations on the gel of DNA ladder bands are shown on the left.

FIG. 3

FIG. 3

Analysis of the presence of an ESS in exon 3 by in vitro splicing assays. (A) The pHS3-ESSV template construct contains the indicated regions of pNL4-3. Shown are 5′ splice site D1 and 3′ splice site A2. The location of the T3 phage polymerase promoter is also shown. The location and the sequence of the putative ESS element are shown with the locations of the mutations underlined. RNA substrates were synthesized from the template as described in Materials and Methods. (B) In vitro splicing of 32P-labeled HIV-1 HS3-ESSV and HS3-ESSVx substrates was analyzed by denaturing PAGE. The positions of the RNA precursor and the spliced product are marked. (C) Ratios of radioactivity in the spliced product compared to that in the unspliced RNA precursor were determined for mutant and wild-type substrates. The results are based on six independent experiments. Standard deviations are shown by error bars.

FIG. 4

FIG. 4

The ESSV sequence acts to inhibit splicing in a heterologous context. (A) Schematic representation of minigene template constructs containing 5′ splice site D1 and 3′ splice site A3 (3). These constructs contain either ESS2, ESSV, or ESSVx. (B) 32P-labeled RNA substrates HS1-ESS2, HS1-ESSV, and HS1-ESSVx were synthesized and RNA splicing was performed as described in Materials and Methods. Products of in vitro splicing of substrates were analyzed on denaturing polyacrylamide gels. The positions of the precursor and the spliced product are marked.

FIG. 5

FIG. 5

Depletion of HeLa cell nuclear extracts with ESSV and ESS2 RNAs removes a common inhibitory cellular factor or factors. In vitro splicing of HS1-ESSV, HS1-ESSVx, or HS1-ESS2 substrates was carried out by use of nondepleted nuclear extracts and nuclear extracts depleted with ESSV- or ESSVx-biotinylated RNAs (A) or ESS2- or ESS2x-biotinylated RNAs (B) immobilized on paramagnetic beads (Beads-RNA) as described in Materials and Methods. The positions of the precursor and the spliced product are marked.

FIG. 6

FIG. 6

Selective depletion of inhibitory proteins in HeLa cell nuclear extracts with ESSV RNA-beads. (A) Samples (50 μg) from nondepleted nuclear extracts and nuclear extracts depleted with either ESSV or ESSVx paramagnetic bead-immobilized RNAs were separated by SDS–10% PAGE and stained with Coomassie blue. Circles on the right indicate differences between the ESSV RNA-beads and the ESSVx RNA-beads. Apparent molecular weights (in thousands) are shown on the left. (B) Western blot analyses of proteins in depleted and nondepleted extracts were carried out with the indicated anti-hnRNP antibodies as described in Materials and Methods. For the analyses with anti-hnRNP A1 and anti-hnRNP B1 antibodies, 20 μg of protein from the depleted or nondepleted extracts was analyzed. For the analysis with anti-hnRNP A2 antibody, 50 μg of protein from the depleted or nondepleted extracts was analyzed. The anti-hnRNP A2 antiserum also detects hnRNP A1. (C) Aliquots (1 to 8 μl) from HeLa cell nuclear extracts (HNE) nondepleted or depleted with either wild-type (ESSV) or mutant (ESSVx) RNA bound to beads were electrophoresed, and Western blot analysis was carried out using anti-hnRNP A1 antibody.

FIG. 7

FIG. 7

Reconstitution of splicing inhibition by addition of hnRNP A/B proteins to depleted extracts. HS1-ESSV and HS1-ESSVx RNA substrates were spliced in ESSV RNA-bead-depleted HeLa cell nuclear extracts in the presence of 900 ng of the indicated hnRNPs. Splicing was also carried out after the addition of the same amounts of control proteins: UP1-GST (two RNA recognition motifs of hnRNP A1 fused to GST) in lanes 11 and 12 and GST alone in lanes 13 and 14. The positions of the precursor and the spliced product are marked.

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References

    1. Adachi A, Gendelman H E, Koenig S, Folks T, Willey R, Rabson A, Martin M A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986;59:284–291. - PMC - PubMed
    1. Aiyar A, Leis J. Modification of the megaprimer method of PCR mutagenesis: improved amplification of the final product. BioTechniques. 1993;14:366–369. - PubMed
    1. Amendt B A, Hesslein D, Chang L-J, Stoltzfus C M. Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of the human immunodeficiency virus type 1. Mol Cell Biol. 1994;14:3960–3970. - PMC - PubMed
    1. Amendt B A, Si Z-H, Stoltzfus C M. Presence of exon splicing silencers within HIV-1 tat exon 2 and tat/rev exon 3: evidence for inhibition mediated by cellular factors. Mol Cell Biol. 1995;15:4606–4615. - PMC - PubMed
    1. Arch R, Wirth K, Hoffman M, Ponta H, Matzku S, Herrlich P, Zoller M. Participation in normal immune response of a splice variant of CD44 that encodes a metastasis-inducing domain. Science. 1992;257:682–685. - PubMed

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