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bioRxiv · 10.1101/2023.08.24.554598

hnRNP A1 induces aberrant CFTR exon 9 splicing via a newly discovered Exonic Splicing Silencer element.

Beaumont, C.·Chou, M.-Y.·Stuani, C.·Buratti, E.·Lukavsky, P. J.
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Abstract

RNA-protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a non-functional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends primarily on variations in the polymorphic (TG)mTn locus upstream of exon 9. At the pre-mRNA level, it generates an extended UG-rich binding site for TDP-43, associated with hnRNP A1 recruitment, and prevention of exon 9 3 splicing site (3ss) recognition. While TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs (RRM1 and RRM2) and a glycine-rich domain, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Thermal Calorimetry (ITC). The affinities are submicromolar and ITC data suggest that the separate RRMs as well as tandem RRMs form 1:1 complexes. NMR titrations reveal that hnRNP A1 interacts with model CTFR 3ss sequences in a fast exchange regime at the NMR timescale. Splicing assays finally show that this hnRNP A1 binding site represents a previously unknown exonic splicing silencer element. Together, our results shed light on the mechanism of aberrant CFTR exon 9 splicing.

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Beaumont, C., Chou, M.-Y., Stuani, C., Buratti, E., Lukavsky, P. J.. 2023-08-24. hnRNP A1 induces aberrant CFTR exon 9 splicing via a newly discovered Exonic Splicing Silencer element.. https://doi.org/10.1101/2023.08.24.554598

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