# hnRNP A1 induces aberrant CFTR exon 9 splicing via a newly discovered ESS element

## TL;DR

This study shows how the protein hnRNP A1 contributes to faulty splicing of the CFTR gene, leading to severe cystic fibrosis.

## Contribution

The discovery of a new exonic splicing silencer element bound by hnRNP A1 in CFTR exon 9.

## Key findings

- hnRNP A1 binds to a newly identified ESS element in CFTR exon 9 with submicromolar affinity.
- NMR and ITC experiments confirm a 1:1 stoichiometry and fast exchange binding regime.
- The binding site acts as an exonic splicing silencer, contributing to exon 9 skipping.

## Abstract

Missplicing of CFTR exon 9 requires TDP 43 binding to UG-rich sequences upstream of the 3′ss and hnRNP A1 binding to an ESS element in CFTR exon 9.

RNA–protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a nonfunctional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends on TDP-43 binding to an extended UG-rich binding site upstream of CFTR exon 9 3′ splicing site (3′ss) and is associated with concomitant hnRNP A1 recruitment. Although TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Titration Calorimetry. The affinities are submicromolar, and Isothermal Titration Calorimetry data suggest complexes with a 1:1 stoichiometry. NMR titrations reveal that hnRNP A1 interacts with model CTFR 3′ss 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.