Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering

Abstract

The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF65) binds the polypyrimidine tract near the 3' splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein-nucleic acid complex. Cocrystals of a U2AF65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 angstroms resolution and contain one complex per asymmetric unit.

Figure 1

(a) Domain map of human U2AF⁶⁵ showing the limits of RRM1 and RRM2. UHM (U2AF homology motif) is a protein-interaction domain (Gozani et al., 1998 ▶) and RS denotes an arginine/serine-rich domain. The RRM1–RRM2 linker is expanded to show a phylogenetic sequence comparison. Residues that are conserved in at least four of the five species shown are indicated in bold. (b) Variants of the linker region (residues 228–258) between human U2AF⁶⁵ RRM1 and RRM2 used for cocrystallization with oligonucleotides. Dashes and numbers in parentheses indicate deleted residues. The ‘d8-linker’ variant is derived from the linker sequence of splicing factor Sex-lethal (‘s-­linker’). The successfully cocrystallized variant is shown in bold.