Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.

2.50
Hdl Handle:
http://hdl.handle.net/10033/620734
Title:
Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.
Authors:
Spadaccini, Roberta; Reidt, Ulrich; Dybkov, Olexandr; Will, Cindy; Frank, Ronald; Stier, Gunter; Corsini, Lorenzo; Wahl, Markus C; Lührmann, Reinhard; Sattler, Michael
Abstract:
The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.
Affiliation:
European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Citation:
Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing. 2006, 12 (3):410-25 RNA
Journal:
RNA (New York, N.Y.)
Issue Date:
Mar-2006
URI:
http://hdl.handle.net/10033/620734
DOI:
10.1261/rna.2271406
PubMed ID:
16495236
Type:
Article
Language:
en
ISSN:
1355-8382
Appears in Collections:
Publications of the research group Chemical Biology (CBIO)

Full metadata record

DC FieldValue Language
dc.contributor.authorSpadaccini, Robertaen
dc.contributor.authorReidt, Ulrichen
dc.contributor.authorDybkov, Olexandren
dc.contributor.authorWill, Cindyen
dc.contributor.authorFrank, Ronalden
dc.contributor.authorStier, Gunteren
dc.contributor.authorCorsini, Lorenzoen
dc.contributor.authorWahl, Markus Cen
dc.contributor.authorLührmann, Reinharden
dc.contributor.authorSattler, Michaelen
dc.date.accessioned2017-01-20T11:50:35Z-
dc.date.available2017-01-20T11:50:35Z-
dc.date.issued2006-03-
dc.identifier.citationBiochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing. 2006, 12 (3):410-25 RNAen
dc.identifier.issn1355-8382-
dc.identifier.pmid16495236-
dc.identifier.doi10.1261/rna.2271406-
dc.identifier.urihttp://hdl.handle.net/10033/620734-
dc.description.abstractThe p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshBase Sequenceen
dc.subject.meshBinding Sitesen
dc.subject.meshElectrophoretic Mobility Shift Assayen
dc.subject.meshHumansen
dc.subject.meshIn Vitro Techniquesen
dc.subject.meshMacromolecular Substancesen
dc.subject.meshModels, Molecularen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshNuclear Magnetic Resonance, Biomolecularen
dc.subject.meshNuclear Proteinsen
dc.subject.meshNucleic Acid Conformationen
dc.subject.meshPhosphoproteinsen
dc.subject.meshProtein Structure, Secondaryen
dc.subject.meshRNA Precursorsen
dc.subject.meshRNA Splicingen
dc.subject.meshRNA Splicing Factorsen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshRibonucleoprotein, U2 Small Nuclearen
dc.subject.meshRibonucleoproteinsen
dc.subject.meshSplicing Factor U2AFen
dc.subject.meshThermodynamicsen
dc.titleBiochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.en
dc.typeArticleen
dc.contributor.departmentEuropean Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.en
dc.identifier.journalRNA (New York, N.Y.)en

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