Intron retention in the alternatively spliced region of RON results from weak 3' splice site recognition

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• The RON gene encodes a tyrosine kinase receptor involved in cell signaling.
• A specific RON isoform, lacking exon 11, is linked to carcinoma invasiveness.
• Intron retention in RON mRNA is observed but its cause is unclear.

Purpose of the Study:

• To investigate the molecular mechanisms underlying intron 10 and 11 retention in RON mRNA.
• To determine if exon sequences or splice site strength are responsible for intron retention.

Main Methods:

• In vitro splicing assays using chimeric pre-mRNAs.
• Systematic testing of exon and splice site sequences.
• UV-crosslinking to identify protein binding factors.
• Analysis of spliceosome assembly intermediates.

Main Results:

• Weak 3' splice site regions adjacent to exons 11 and 12 were identified as the cause of intron retention.
• Exon sequences modulating exon 11 skipping were not limiting.
• hnRNP F/H binding occurred 5' of weak splice sites but did not inhibit splicing.
• Failure of splicing was linked to weak binding of U2AF65, stalling spliceosome assembly.

Conclusions:

• Weak 3' splice sites and impaired U2AF65 binding are the primary drivers of intron retention in RON mRNA.
• This splicing defect may contribute to the invasive phenotype observed in carcinomas.
• Further research is needed to understand in vivo mechanisms facilitating U2AF65 binding.