SRp30c is a repressor of 3' splice site utilization

Mol Cell Biol. 2002 Jun;22(12):4001-10. doi: 10.1128/mcb.22.12.4001-4010.2002.

Abstract

Several intron elements influence exon 7B skipping in the mammalian hnRNP A1 pre-mRNA. We have shown previously that the 38-nucleotide CE9 element located in the intron separating alternative exon 7B from exon 8 can repress the use of a downstream 3' splice site. The ability of CE9 to act on heterologous substrates, combined with the results of competition and gel shift assays, indicates that the activity of CE9 is mediated by a trans-acting factor. UV cross-linking analysis revealed the specific association of a 25-kDa nuclear protein with CE9. Using RNA affinity chromatography, we isolated a 25-kDa protein that binds to CE9 RNA. This protein corresponds to SRp30c. Consistent with a role for SRp30c in the activity of CE9, recombinant SRp30c interacts specifically with CE9 and can promote splicing repression in vitro in a CE9-dependent manner. The closest homologue of SRp30c, ASF/SF2, does not bind to CE9 and does not repress splicing even when the intronic SRp30c binding sites are replaced with high-affinity ASF/SF2 binding sites. Only the first 7 nucleotides of CE9 are sufficient for binding to SRp30c, and mutations that abolish binding also prevent repression. Our results indicate that SRp30c can function as a repressor of 3' splice site utilization and suggest that the SRp30c-CE9 interaction may contribute to the control of hnRNP A1 alternative splicing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Binding Sites
  • Exons
  • HeLa Cells
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B*
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Humans
  • Introns
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • RNA Precursors / metabolism
  • RNA Splice Sites*
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Regulatory Sequences, Nucleic Acid
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism
  • Serine-Arginine Splicing Factors

Substances

  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Nuclear Proteins
  • Phosphoproteins
  • RNA Precursors
  • RNA Splice Sites
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Ribonucleoproteins
  • Serine-Arginine Splicing Factors