Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene.

Jana Kralovicova,Pedro M.D Moreno,Igor Vorechovsky,Ana Paula Pêgo,Nicholas C.P Cross

doi:10.1089/nat.2016.0635

Abstract

ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs.

Highlights

Eukaryotic genes contain intervening sequences or introns that must be removed by a large and highly dynamic RNA protein complex termed the spliceosome to ensure accurate protein synthesis [1]
We have shown for the first time that a chitosan-based delivery system for intronic SSOs can repress an NSE (Fig. 3)
Our results demonstrate the first transposed elements that promote or repress inclusion of an NSE in mature transcripts (Fig. 1)

Summary

Introduction

Eukaryotic genes contain intervening sequences or introns that must be removed by a large and highly dynamic RNA protein complex termed the spliceosome to ensure accurate protein synthesis [1]. Once regarded a selfish or junk DNA, introns are recognized as critical functional components of eukaryotic genes that enhance gene expression and regulate alternative RNA processing, mRNA export, and RNA surveillance [2,3] They are an important source of new gene-coding and regulatory sequences [1,4,5] and noncoding RNAs, including microRNAs and circular RNAs [6,7]. Their removal process is tightly coupled with transcription, mRNA export, and translation, with most human introns eliminated from pre-mRNA cotranscriptionally [8]. Their potential as targets for nucleic acid therapy is only beginning to be unleashed

Methods

Results

Conclusion