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

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• Pre-messenger RNA (pre-mRNA) alternative splicing generates diverse transcript isoforms.
• U2AF2 protein binding to 3' splice site polypyrimidine tracts (PPTs) is crucial for splice acceptor determination.
• The precise mechanism of U2AF2's PPT recognition is not fully understood.

Purpose of the Study:

• To investigate the molecular mechanism by which U2AF2 distinguishes between various PPTs.
• To identify key structural motifs in U2AF2 involved in PPT recognition and alternative splicing modulation.

Main Methods:

• In vivo amino acid scanning mutagenesis of a conserved α-helix in U2AF2's RNA recognition motif 1 (RRM1).
• Genome-wide alternative splicing analysis in Caenorhabditis elegans.
• Structural modeling and molecular dynamics simulations of U2AF2-RNA interactions.
• Mutagenesis of PPT nucleotides in transgenic splicing reporters.

Main Results:

• A conserved α-helix in U2AF2's RRM1 was identified as a key motif for PPT recognition.
• Amino acid substitutions within this helix dynamically modulated genome-wide alternative splicing in C. elegans.
• Structural modeling and simulations predicted that specific residue sidechains induce flexible twists, enabling induced-fit binding to PPT nucleotides.
• Mutations in PPT nucleotides confirmed the predicted binding interactions and alternative splicing outcomes.

Conclusions:

• U2AF2 utilizes a novel structure-function mechanism involving a conserved N-terminal α-helix to modulate alternative splicing.
• The flexibility and specific sidechain interactions within the α-helix allow U2AF2 to adjust its binding to diverse PPT sequences.
• This study provides critical insights into the regulation of alternative splicing by U2AF2.