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

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

Jana Kralovicova et al. Nucleic Acid Ther. 2016 Dec.

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.

Keywords: ATM; alternative splicing; antisense oligonucleotides; lymphoid cancer; nanoparticles; nonsense-mediated RNA decay; transposon.

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Conflict of interest statement

Author Disclosure Statement A part of this work is subject to a UK patent application by the University of Southampton.

Figures

<b>FIG. 1.</b>
FIG. 1.

Identification of transposed elements in ATM intron 28 that influence NSE activation. (A) Location of transposed elements in intron 28 and schematics of NSE activation. Canonical exons [54] are shown as gray boxes, the NSE as a white box, introns flanking the NSE as horizontal lines, and their splicing by dotted lines. Deletions (numbered 1–6) of transposed elements are shown as horizontal white rectangles; UC, a unique sequence lacking recognizable transposons. Deletion numbers correspond to lanes in (B). RT-PCR primers are denoted by black arrows. A scale is at the top. The NSE sequence is boxed in the lower panel. Asterisk denotes the C/T variant rs609261 located at the NSE 3′ss; rs4988000 (not shown) is 64 bp downstream of the NSE 5′ss. (B) Deletion of antisense Alu and MER51 alters NSE inclusion levels. WT and mutated constructs [designated 1–6 in (A)] were transiently transfected into HEK293 cells (mock) depleted of U2AF35. NSE+/−, RNA products with/without NSE. Columns represent mean NSE inclusion (%), error bars are SDs of two independent transfection experiments. Asterisks denote P values <0.01 for comparisons with the WT. 5′ss, 5′ splice site; ATM, ataxia-telangiectasia, mutated; NSE, NMD switch exon; RT-PCR, reverse transcription polymerase chain reaction; SDs, standard deviations; WT, wild-type.

<b>FIG. 2.</b>
FIG. 2.

Identification of intronic SSOs that activate or repress NSE. (A) Location of tested SSOs in intron 28 relative to transposed elements (for legend, see Fig. 1A). The branch point sequence (GGCTG

A

T; branch point adenosine is underlined) of NSE is denoted by a vertical arrowhead. (B) Intronic SSOs that alter NSE inclusion in exogenous transcripts. SSOs are at the bottom. Multiple controls are boxed. SSO sequences are in Table 2. The average NSE inclusion in controls is denoted by a dotted line, error bars are SDs of two independent transfection experiments. Columns represent mean NSE inclusion levels, asterisks show significant P values. (C) SSOs targeting single-stranded regions tended to repress endogenous NSE. r, Pearson correlation coefficient. The P value is in brackets. SSOs, splice-switching oligonucleotides.

<b>FIG. 3.</b>
FIG. 3.

TMC-SA-assisted delivery of SSO-NSE3 to human cell lines leads to NSE repression. (A) NSE inclusion in HEK293 cells is inhibited upon exposure of SSO-NSE3/TMC-SA nanocomplexes. Sc, a scrambled control with the same modification. M, size marker. Error bars denote SDs of two transfection experiments. P values are shown at the top for the indicated comparisons. (B) NSE repression in VAVY cells exposed to the SSO-NSE3/TMC-SA nanocomplexes. TMC-SA, stearylated trimethylated chitosan.

<b>FIG. 4.</b>
FIG. 4.

Inverted repeats in the MER51 element of ATM intron 28. The alignment was carried out by RepeatMasker [35]. v, transversions; i, transitions. Putative purine-rich loops (highlighted in gray) are flanked by inverted repeats (underlined) that may form stable base-pairing interactions during transcription. The long terminal repeat homology region originally described for the MER51 family [49] is in italics. The aligned segment corresponds to deletion 4 shown in Fig. 1A. The MER51A consensus sequence is in the antisense orientation.

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