Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60

Lorenzo Corsini, Michael Hothorn, Gunter Stier, Vladimir Rybin, Klaus Scheffzek, Toby J. Gibson, Michael Sattler

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

Abstract

PUF60 is an essential splicing factor functionally related and homologous to U2AF 65. Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimer-ization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 Å resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF 65, and SF3b155. Compared with U2AF 65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF 65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.

Original languageEnglish
Pages (from-to)630-639
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number1
DOIs
StatePublished - 2 Jan 2009

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