High-throughput sequencing of
hematologic malignancies and other
cancers has revealed recurrent mis-sense mutations of genes encoding
pre-mRNA splicing factors. The essential
splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with
leukemia or solid
tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2
proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent
RNA-binding affinity of the N196K-substituted
protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the
RNA phosphodiester could explain the decreased
RNA-binding affinity of the G301D-substituted
protein. We found that expression of the G301D-substituted U2AF2
protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in
cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.