Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms

Area of Science:

• Molecular Biology
• RNA Splicing
• Gene Regulation

Background:

• Mechanisms of splicing regulation via non-canonical splice junction processing are not well understood.
• Alternative pre-mRNA splicing plays a crucial role in generating protein diversity.

Purpose of the Study:

• To investigate the regulatory mechanisms of non-canonical 3' splice site selection in the RBFOX2 gene.
• To characterize the functional differences between canonical and non-canonical RBFOX2 splicing isoforms.

Main Methods:

• Identification of RBFOX2 splicing isoforms using alternative 3' splice site selection.
• Analysis of subcellular localization (cytoplasmic vs. nuclear) of RBFOX2 isoforms.
• Investigation of stress granule localization upon sodium arsenite treatment.
• Minigene assays to assess the role of cis-regulatory elements in non-canonical splice site selection.

Main Results:

• Two RBFOX2 isoforms, RBFOX2-C. (canonical) and RBFOX2-N.C. (non-canonical), were identified.
• RBFOX2-N.C. utilizes a non-canonical 3' splice GG acceptor sequence.
• RBFOX2-N.C. exhibits nuclear localization, distinct from the cytoplasmic localization of RBFOX2-C.
• RBFOX2-C. shows stronger localization to stress granules than RBFOX2-N.C. under stress conditions.
• The non-canonical 3' splice GG sequence and adjacent suboptimal branch point site region are critical for RBFOX2-N.C. expression.

Conclusions:

• A regulatory mechanism for non-canonical 3' splice selection in the RBFOX2 gene was elucidated.
• Findings provide a foundation for understanding alternative pre-mRNA splicing regulation through non-canonical processing.
• Differential localization and stress granule incorporation highlight functional divergence between RBFOX2 isoforms.