title: Solution structure of the HIV-1 exon splicing silencer 3.
abstract: Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.8±1.1 nM) complex with ESS3 via site-specific interactions with the loop.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Levengood
authors.forename: Carrie
authors.initials: C
authors.lastname: Rollins
authors.forename: Clay H J
authors.initials: CH
authors.lastname: Mishler
authors.forename: Charles A
authors.initials: CA
authors.lastname: Johnson
authors.forename: Grace
authors.initials: G
authors.lastname: Miner
authors.forename: Prashant
authors.initials: P
authors.lastname: Rajan
authors.forename: Brent M
authors.initials: BM
authors.lastname: Znosko
authors.forename: Blanton S
authors.initials: BS
authors.lastname: Tolbert
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D005091:Exons / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D022821:RNA Splice Sites / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D037941:Silencer Elements, Transcriptional / Q000235:genetics* / Q000502:physiology; D012996:Solutions / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; D022821:RNA Splice Sites; D012367:RNA, Viral; D012996:Solutions