Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

Simon Braun,Mariela Cortés-López,Stefan Legewie,Markus Seiler,Kathi Zarnack,Anke Busch,Nuno L Barbosa-Morais,Mihaela Enculescu,Samarth T Setty,Stefanie Ebersberger,Laura Schulz,F X Reymond Sutandy,Bernardo P De Almeida,Julian König

doi:10.1038/s41467-018-05748-7

Abstract

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

Highlights

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer
To systematically study the cis-regulatory sequence elements that control Recepteur d’origine nantais (RON) alternative splicing, we designed an in vivo screening approach based on random mutagenesis of a splicing reporter minigene (Fig. 1a)
Mutations in a known cis-regulatory element led to increased RON exon 11 skipping as reported previously[7] (Supplementary Fig. 1c)

Summary

Introduction

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Splicing is catalysed by the spliceosome, a multi-subunit complex that recognises the 5′ and 3′ splice sites and flanking sequence elements in the pre-mRNA The latter include the polypyrimidine tract (Py-tract) and the branch point upstream of each exon[10]. Heterogeneous nuclear ribonucleoprotein H (HNRNPH; collectively referring to HNRNPH1 and its close paralogue HNRNPH2 which are 96% identical at the amino acid level28) was found to repress RON exon 11 inclusion via binding within the alternative exon. While these studies suggested that RON splicing is heavily regulated, most cis-regulatory elements remain unknown

Methods

Results

Conclusion