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

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54 Scopus citations


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
Issue number1
StatePublished - 2 Jan 2009


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