Selection of alternative 5' splice sites

Role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

I. C. Eperon, O. V. Makarova, Akira Maeda, S. H. Munroe, J. F. Caceres, D. G. Hayward, A. R. Krainer

Research output: Contribution to journalArticle

134 Citations (Scopus)

Abstract

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

Original languageEnglish
Pages (from-to)8303-8318
Number of pages16
JournalMolecular and Cellular Biology
Volume20
Issue number22
DOIs
Publication statusPublished - 11-11-2000

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U1 Small Nuclear Ribonucleoproteins
RNA Splice Sites
RNA Precursors
hnRNP A1
Protein Splicing
Introns

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Eperon, I. C. ; Makarova, O. V. ; Maeda, Akira ; Munroe, S. H. ; Caceres, J. F. ; Hayward, D. G. ; Krainer, A. R. / Selection of alternative 5' splice sites : Role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1. In: Molecular and Cellular Biology. 2000 ; Vol. 20, No. 22. pp. 8303-8318.
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abstract = "The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.",
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Selection of alternative 5' splice sites : Role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1. / Eperon, I. C.; Makarova, O. V.; Maeda, Akira; Munroe, S. H.; Caceres, J. F.; Hayward, D. G.; Krainer, A. R.

In: Molecular and Cellular Biology, Vol. 20, No. 22, 11.11.2000, p. 8303-8318.

Research output: Contribution to journalArticle

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T2 - Role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

AU - Eperon, I. C.

AU - Makarova, O. V.

AU - Maeda, Akira

AU - Munroe, S. H.

AU - Caceres, J. F.

AU - Hayward, D. G.

AU - Krainer, A. R.

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N2 - The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

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