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hnRNP A1 controls HIV-1 mRNA splicing th ... a conserved secondary structure
hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure
7333
The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.
Damgaard CK, Tange TO, Kjems J
RNA (New York, N.Y.)
2002-11-01 00:00
8
11
1401-15
Base Sequence,Conserved Sequence,Electrophoretic Mobility Shift Assay,Exons,Gene Products, tat,Gene Silencing,Glutathione Transferase,HIV-1,Hela Cells,Heterogeneous-Nuclear Ribonucleoprotein Group A-B,Humans,Introns,Molecular Sequence Data,Nucleic Acid Conformation,Protein Footprinting,RNA Precursors,RNA Splicing,RNA, Messenger,RNA, Viral,Recombinant Fusion Proteins,Regulatory Sequences, Nucleic Acid,Ribonucleoprotein, U2 Small Nuclear,Spliceosomes,Gene Products, tat,Heterogeneous-Nuclear Ribonucleoprotein Group A-B,RNA Precursors,RNA, Messenger,RNA, Viral,Recombinant Fusion Proteins,Ribonucleoprotein, U2 Small Nuclear,hnRNP A1,Glutathione Transferase
Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
RNA
1355-8382
0
False
12458794
