Loss-of-function uORF mutations in human malignancies

Julia Schulz,Martin Neuenschwander,Oliver Klaas,Wolfgang E Berdel,Carsten Denkert,Miguel A Andrade-Navarro,Per-Ulf Tunn,Tabea Kischka,Jens P Von Kries,Iduna Fichtner,Klaus Wethmar,Peter M Schlag,Achim Leutz,Richard Ratei,Wojciech Makalowski,Nancy Mah,Esmeralda Castaños-Vélez

doi:10.1038/s41598-018-19201-8

Abstract

Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. Translational activation of proto-oncogenes through loss-of-uORF mutations has been demonstrated, yet a systematic search for cancer-associated genetic alterations in uORFs is lacking. Here, we applied a PCR-based, multiplex identifier-tagged deep sequencing approach to screen 404 uORF translation initiation sites of 83 human tyrosine kinases and 49 other proto-oncogenes in 308 human malignancies. We identified loss-of-function uORF mutations in EPHB1 in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma. Both mutations were associated with enhanced translation, suggesting that loss-of-uORF-mediated translational induction of the downstream main protein coding sequence may have contributed to carcinogenesis. Computational analysis of whole exome sequencing datasets of 464 colon adenocarcinomas subsequently revealed another 53 non-recurrent somatic mutations functionally deleting 22 uORF initiation and 31 uORF termination codons, respectively. These data provide evidence for somatic mutations affecting uORF initiation and termination codons in human cancer. The insufficient coverage of uORF regions in current whole exome sequencing datasets demands for future genome-wide analyses to ultimately define the contribution of uORF-mediated translational deregulation in oncogenesis.

Highlights

Ribosome profiling revealed widespread translational activity at upstream open reading frames and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression
We identified loss-of-function uORF mutations in ephrin receptor B1 (EPHB1) in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma
To define the uORF-target set, we mapped genomic positions of all uORF initiation codons in 132 cancer-associated genes (Fig. 1B, Supplementary Table 1), comprising 83 human tyrosine kinases (TKs)[5, 46] validated proto-oncogenes overexpressed or amplified in human cancer[18], and three candidate genes post-transcriptionally induced in cancer cell lines[19]

Summary

Introduction

Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. We identified loss-of-function uORF mutations in EPHB1 in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma Both mutations were associated with enhanced translation, suggesting that loss-of-uORF-mediated translational induction of the downstream main protein coding sequence may have contributed to carcinogenesis. Computational analysis of whole exome sequencing datasets of 464 colon adenocarcinomas subsequently revealed another 53 non-recurrent somatic mutations functionally deleting 22 uORF initiation and 31 uORF termination codons, respectively. Ribosome profiling and numerous observations on individual transcripts characterized upstream open reading frames (uORFs) as repressive cis-regulatory elements, constitutively reducing translation rates of downstream main protein coding sequences (CDSs)[1,2,3,4]. Despite a mostly repressive effect on downstream translation, specific uORFs mediate a variety of molecular responses, including the translational induction of key regulatory proteins during the integrated stress response[9,10], the adjustment of protein levels in response to uORF-specific co-regulators[11], and the control of balanced protein isoform expression from a single transcript[12]

Methods

Results

Conclusion