Genome-wide modulation of alternative splicing by a predicted alpha helix in U2AF2

Pre-mRNA alternative splicing generates diverse transcript isoforms from the same pre-mRNAs. The binding of 3' splice site polypyrimidine tracts (PPTs) by U2AF2 is an essential early step in determining the final splice acceptors. However, the mechanism by which U2AF2 distinguishes various PPTs remains to be fully understood. Here, we provide molecular genetic evidence that a conserved α-helix at the N-terminus of U2AF2 RNA recognition motif 1 (RRM1) may be a key motif that modulates PPT recognition. In vivo amino acid scanning of a conserved residue in the helix can dynamically modulate genome-wide alternative splicing correlated with specific PPT nucleotides in Caenorhabditis elegans. Structural modeling of the helix and molecular dynamics simulation of C. elegans U2AF2 binding to a 3' splice site RNA predicted that sidechains of two conserved residues generate flexible twists within the helix, adjusting the orientations of the nucleotide-contacting sidechains to enable an induced-fit binding to PPT nucleotides. Consistent with this prediction, mutagenesis of key PPT nucleotides in transgenic splicing reporters elicited alternative splicing events aligned with the structural models. Together, our findings support a novel structure-function mechanism by which U2AF2 modulates widespread alternative splicing events.