The Rbfox1/LASR complex controls alternative pre-mRNA splicing by recognition of multipart RNA regulatory modules.

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Rbfox proteins are key regulators of alternative pre-mRNA splicing, binding to GCAUG RNA elements.
• In the nucleus, Rbfox associates with the large assembly of splicing regulators (LASR) complex.
• The precise mechanism of Rbfox/LASR complex cooperation in RNA binding and splicing regulation is not fully understood.

Purpose of the Study:

• To map the transcriptome-wide RNA binding sites of the Rbfox1/LASR complex.
• To elucidate how Rbfox and LASR subunits cooperate to recognize RNA motifs and regulate splicing.
• To investigate the functional significance of recognizing multipart modules of RNA motifs.

Main Methods:

• Nuclease protection assay to determine RNA footprints of Rbfox1/LASR on nascent RNA.
• Analysis of a mutant Rbfox1(F125A) to differentiate Rbfox1 and LASR subunit binding sites.
• Splicing analyses and minigene experiments to assess regulatory effects.

Main Results:

• Rbfox1/LASR binds not only GCAUG but also motifs recognized by LASR subunits (hnRNPs M, H/F, C, and Matrin3).
• These RNA elements are often found in tandem, forming multipart regulatory modules.
• A mutant Rbfox1(F125A) lost GCAUG binding but retained LASR subunit motif binding, confirming distinct roles.
• Rbfox regulates splicing through both GCAUG and LASR subunit binding sites, demonstrating combinatorial control.

Conclusions:

• The Rbfox/LASR complex recognizes diverse, tandemly arranged RNA motifs, expanding the understanding of splicing regulation.
• This complex decodes combinatorial splicing signals by binding groups of RNA elements, enabling precise control over alternative splicing.
• Findings highlight the sophisticated mechanisms by which multi-protein complexes achieve specific gene expression regulation.