U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing.

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• Splicing factor 3b subunit 1 (SF3B1) is vital for spliceosomes and frequently mutated in cancers.
• The role of wild-type SF3B1 in regulating alternative splicing (AS) remains largely unknown.
• Cancer-associated SF3B1 mutations are linked to aberrant splicing.

Purpose of the Study:

• To investigate the genome-wide alternative splicing regulatory functions of wild-type SF3B1.
• To identify specific pre-mRNA targets regulated by SF3B1.
• To elucidate the molecular mechanisms underlying SF3B1's role in alternative splicing.

Main Methods:

• RNA sequencing (RNA-seq) to analyze genome-wide alternative splicing in SF3B1 knockdown cells.
• Reverse transcription PCR (RT-PCR) to validate splicing changes in various cell lines and patient cells.
• Site-directed mutagenesis to assess the importance of SF3B1 domains and polypyrimidine tract (PPT) sequences.

Main Results:

• SF3B1 knockdown significantly altered genome-wide alternative splicing, including skipped exons (SEs), alternative 5'/3' splice-site selection, mutually exclusive exons (MXE), and intron retention (RI).
• Survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was identified as a key regulatory target of SF3B1.
• The U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 is essential for its function in SMN2 exon 7 splicing, highlighting the importance of U2AF65 affinity to the polypyrimidine tract.

Conclusions:

• Wild-type SF3B1 plays a significant role in regulating alternative splicing, particularly for SMN2 exon 7.
• SF3B1's interaction with U2AF65, mediated by the polypyrimidine tract, is critical for its splicing regulatory function.
• These findings provide insights into SF3B1's broader functions in gene expression and potential implications for diseases like spinal muscular atrophy.