hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B.
Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.
Expert-Bezançon
Alain
A
Centre de Génétique Moléculaire, CNRS UPR 2167, Laboratoire Propre Associé à l'Université Pierre et Marie Curie, 91198 Gif-sur-Yvette, France.
Sureau
Alain
A
Durosay
Patrice
P
Salesse
Roland
R
Groeneveld
Herman
H
Lecaer
Jean Pierre
JP
Marie
Joëlle
J
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005091:Exons; D020868:Gene Silencing; D017930:Genes, Reporter; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000502:physiology*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry; D006801:Humans; D007438:Introns; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012322:RNA Precursors; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D012261:Ribonucleoproteins / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic; D014162:Transfection; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014335:Tropomyosin; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D012313:RNA