uORF-targeting steric block antisense oligonucleotides do not reproducibly activate RNASEH1 expression

Abstract

AbstractUpstream open reading frames (uORFs) arecis-regulatory motifs that are predicted to occur in the 5ʹ untranslated region (UTR) of the majority of human protein-coding transcripts. uORFs are typically associated with repression of the downstream primary open reading frame (pORF) at either the level of translation, or by promoting mRNA turnover via the nonsense-mediated decay pathway. Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was recently reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons in order to inhibit translation initiation, and thereby disrupt uORF-mediated gene regulation. Given the relative maturity of the oligonucleotide field, such a uORF blocking mechanism might have widespread therapeutic utility. Here, we re-synthesised three of the most potent ASOs targeting theRNASEH1uORF described in the study by Lianget al. and investigated their potential for RNASEH1 protein upregulation. No upregulation (of endogenous or reporter protein expression) was observed with any of the oligonucleotides tested at doses ranging from 25 nM to 300 nM. Conversely, we observed downregulation of expression in some instances, consistent with well-established mechanisms of blocking ribosome procession. Experiments were performed using multiple transfection protocol setups, with care taken to replicate the conditions of the original study. Transfection efficiency was confirmed using aMALAT1-targeting gapmer ASO as a positive control. We conclude that previously-describedRNASEH1uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.