The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The U2AF small subunit (U2AF(SM)) plays a key role in 3' splice site recognition during RNA splicing.
• U2AF(SM) is more conserved than its partner but less studied.
• Understanding U2AF(SM) function is critical for comprehending splicing mechanisms.

Purpose of the Study:

• To investigate the functional conservation of U2AF(SM) between Schizosaccharomyces pombe (yeast) and humans.
• To identify critical domains and residues within U2AF(SM) essential for its function.
• To elucidate the roles of U2AF(SM) in RNA binding and large-subunit interaction.

Main Methods:

• In vivo functional complementation assays using human U2AF(SM) in S. pombe.
• Comprehensive mutational analysis of U2AF(SM) domains.
• Two- and three-hybrid analyses to assess protein interactions and RNA binding.

Main Results:

• Human U2AF(SM) can functionally replace yeast U2AF(SM) in vivo, confirming extensive functional conservation.
• All three conserved domains of U2AF(SM) are essential for cell viability.
• Specific amino acids involved in RNA recognition are critical, while a tryptophan in the pseudo-RNA recognition motif is dispensable for large-subunit interaction.
• Mutations primarily affect RNA binding rather than large-subunit interactions.

Conclusions:

• U2AF(SM) function is remarkably conserved across species, highlighting its fundamental role in splicing.
• The study provides detailed insights into the structure-function relationships of U2AF(SM) domains.
• RNA binding is a more sensitive indicator of U2AF(SM) dysfunction than large-subunit interaction defects.