Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA.
Splicing of the chicken beta-tropomyosin exon 6A is stimulated, both in vivo and in vitro, by an intronic pyrimidine-rich element (S4) located 37 nucleotides downstream of exon 6A. Several pyrimidine-rich sequences are able to substitute for the natural S4 enhancer with various stimulatory effects. We show that the different enhancer sequences recruit U1 small nuclear ribonucleoprotein (SnRNP) to the exon 6A 5' splice site, with an efficiency that correlates with the splicing activation. By using RNA affinity and two-dimensional gel electrophoresis, we characterized several proteins that bind to the different enhancer sequences. Heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP I (polypyrimidine track-binding protein, PTB) exhibit a higher level of interaction with the strong enhancer sequences (S4) than with the weakest enhancers. Functional analysis shows that hnRNP K is a component of the enhancer complex that promotes exon 6A splicing through the wild-type S4 sequence. The addition of recombinant hnRNP K to nuclear extracts preincubated with poly(rC) RNA competitor completely restores splicing efficiency to the original level. hnRNP I (PTB) was also found associated with the strong enhancer sequences. Its function in the splicing of exon 6A is discussed.
Expert-Bezançon
Alain
A
Centre de Génétique Moléculaire, CNRS, F-91190 Gif-sur-Yvette, France.
Le Caer
Jean Pierre
JP
Marie
Joëlle
J
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011066:Poly C / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D014335:Tropomyosin / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014335:Tropomyosin; D011066:Poly C; D012313:RNA