Degenerate minigene library analysis enables identification of altered branch point utilization by mutant splicing...

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• Mutations in the splicing factor SF3B1 are linked to various cancers.
• These mutations cause altered 3' splice site (3'SS) selection, but the underlying mechanisms are not fully understood.
• SF3B1 is crucial for stabilizing U2 snRNP and branch point (BP) interactions during pre-mRNA splicing.

Purpose of the Study:

• To quantitatively determine branch point (BP) utilization by SF3B1-mutant spliceosomes.
• To elucidate the molecular mechanisms by which SF3B1 mutations contribute to oncogenesis through altered splicing.

Main Methods:

• Overexpression of SF3B1 variants in human cells.
• CRISPR/Cas9 genome editing to create isogenic murine embryonic stem cells with SF3B1 mutations (K700E).
• Utilized a large-scale synthetic minigene library to compare BP usage between SF3B1-mutant and wild-type spliceosomes.

Main Results:

• SF3B1-mutant spliceosomes (SF3B1K700E) exhibit increased utilization of non-canonical BP sequences compared to wild-type (SF3B1WT).
• This preference for alternative BP sequences was confirmed through minigene splicing assays.
• Demonstrated that mutant SF3B1 can stabilize weaker U2-BP interactions, enabling the use of non-consensus BP sequences.

Conclusions:

• SF3B1 mutations promote oncogenesis by altering spliceosome's ability to select branch points.
• Mutant SF3B1 stabilizes suboptimal U2-BP interactions, leading to the use of alternative BP sequences and aberrant splicing.
• Findings provide a mechanistic link between SF3B1 mutations, altered splicing, and cancer development.