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

doi:10.1074/jbc.m805395200

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 dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms 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, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.

Highlights

Pre-mRNA splicing is a stepwise process initiated by the recognition of sequence elements at the splice site by specific splicing factors [1]
Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-U2AF homology motif (UHM) binds to UHM ligand motifs (ULM) sequences in the splicing factors SF1, U2AF65, and SF3b155
The branch point sequence is recognized by splicing factor SF1 [2, 3], whereas the polypyrimidine tract and the 3Ј splice site AG-dinucleotide are bound by the heterodimer U2AF65-U2AF35 (4 –7)

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation—Recombinant Puf60-UHM (residues 460 –559, wild type, and mutants), thioredoxin-Puf60-UHM, SPF45-UHM[301– 401], U2AF35-UHM (residues 38 –152), and U2AF65-UHM (residues 369 – 475) were expressed from modified pET9d vectors with a noncleavable N-terminal His tag. 13C- and/or 15N-labeled proteins were expressed in minimal (M9) medium supplemented with 13C-D-glucose and/or 15NH4Cl. NMR samples were concentrated to 0.3–1.0 mM in 20 mM Na2PO4 buffer (pH 6.8), 150 mM NaCl, and 5 mM ␤-mercaptoethanol. Crystallization and Data Collection—For crystallization, the chimeric thioredoxin-Puf60(460 –559) fusion protein was concentrated to about 70 mg/ml in 20 mM Tris (pH 7.0), 150 mM NaCl, 5 mM ␤-mercaptoethanol. The eight UHM domains in the unit cell of the crystal structure can be superimposed onto a reported solution structure of Puf60-UHM (Protein Data Bank code 2DNY) with root mean square deviations of 0.9 –1.1 Å over 90 of 100 C␣ atoms. GST Pulldown Experiments—GST-tagged ULMs (1 nmol) were mixed with 3 nmol of His6-tagged UHMs in 150 ␮l of phosphate-buffered saline supplemented with 2 mM ␤-mercaptoethanol and 0.1% (w/v) Igepal CA-630 at 22 °C and mixed vigorously for 1 h. Western blotting was carried out with ␣-Puf antibody (Abcam 22819)

RESULTS

Space group Unit cell dimensions

Molar Ratio

DISCUSSION

HEAT repeats