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

Abstract

Upstream open reading frames (uORFs) are cis-regulatory motifs that are predicted to occur in the 5' UTRs of the majority of human protein-coding transcripts and are typically associated with translational repression of the downstream primary open reading frame (pORF). Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was previously reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons 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-synthesized three of the most potent ASOs targeting the RNASEH1 uORF described in a study by Liang et al. and investigated their potential for RNASEH1 protein upregulation, with care taken to replicate the conditions of the original study. No upregulation (of endogenous or reporter protein expression) was observed with any of the oligonucleotides tested at doses ranging from 25 to 300 nM. Conversely, we observed downregulation of expression in some instances. We conclude that previously described RNASEH1 uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.

Keywords: MT: Oligonucleotides: Therapies and Applications; RNASEH1; antisense oligonucleotides; steric block ASO; uORF; upstream open reading frame.

Graphical abstract

Figure 1

RNASEH1 uORF-targeting steric block antisense oligonucleotides (A) Schematic of ASO sequences and chemistries used in this study. (B) Sequences of on-target RNASEH1 uORF-targeting ASOs and their scrambled control (Ctrl) sequences for the 16mer and 18mer variants. (C) Chemical structures of key oligonucleotide modifications incorporated into ASOs used in this study.

Figure 2

Validation of RNASEH1 uORF activity (A) Schematic of the RNASEH1 transcript showing the position, sequence, and translation of the uORF and binding locations for the uORF-targeting ASOs. Aggregated Ribo-Seq/RNA-Seq data are overlaid, providing evidence of uORF translation. The uORF is highlighted in yellow, and the start codons are highlighted in bold and underlined. TSS, transcription start site. HEK293T cells were transfected with plasmids expressing either the wild-type (WT) RNASEH1 5′ UTR dual luciferase reporter (DLR) construct, or a mutant construct (TTG) in which the predicted uORF was disrupted by mutation of the uORF start codon. Treated cultures were analyzed after 24 h for (B) DLR assay or (C) RT-qPCR. Values are mean + SD (n = 3 independent experiments) and were scaled such that the mean of the WT control group was returned to a value of 1. Statistical significance was determined by unpaired Student’s t test; ∗∗∗∗p < 0.0001.

Figure 3

uORF-targeting steric block ASOs do not increase RNASEH1 protein expression at 4, 8, and 12 h post-transfection HeLa cells were transfected with 100 nM ASOs or matched chemistry controls and cells harvested at (A) 4 h, (B) 8 h, or (C) 12 h post-transfection. RNASEH1 protein was quantified by Jess capillary western blot. Vinculin (VCL) was used as a loading control. Representative blots are shown together with histograms of protein quantification. The values of untreated control samples are indicated by dotted lines (scaled to a value of 1). A gapmer targeting MALAT1 was included as a positive control for transfection, which is not expected to influence RNASEH1 expression. Values are mean + SD. Statistical significance was assessed by paired Student’s t test between each treatment and its respective control ASO; ∗p < 0.05; n = 4 completely independent experiments.

Figure 4

uORF-targeting steric block ASOs do not increase RNASEH1 protein expression at 24, 48, and 72 h post-transfection HeLa cells were transfected with 100 nM ASOs or matched chemistry controls and cells harvested at (A) 24 h, (B) 48 h, or (C) 72 h post-transfection. RNASEH1 protein was quantified by Jess capillary western blot. VCL was used as a loading control. Representative blots are shown together with histograms of protein quantification. The value of untreated control samples is indicated by the dotted line (scaled to a value of 1). A gapmer targeting MALAT1 was included as a positive control for transfection, which is not expected to influence RNASEH1 expression. Values are mean + SD. Statistical significance was assessed by paired Student’s t test between each treatment and its respective control ASO (no significant changes detected); n = 4 completely independent experiments.

Figure 5

uORF-targeting steric block ASOs do not increase RNASEH1 protein expression, regardless of dose HeLa cells were transfected with ASOs at concentrations as indicated and protein harvested after 48 h for (A) PO-2OMe, (B) PS-MOE, and (C) PS-2OMe nucleic acid chemistries. RNASEH1 protein was quantified by Jess capillary western blot. VCL was used as a loading control. Representative blots are shown together with histograms of protein quantification. The value of untreated control samples is indicated by the dotted line (scaled to a value of 1). (D) Cells were transfected with a gapmer targeting MALAT1 (100 nM) in parallel as a positive control for transfection. MALAT1 transcript levels were determined by RT-qPCR and normalized to RPL10 expression. Values are mean + SD. Statistical significance for protein data were assessed by paired Student’s t test within each oligonucleotide dose. RT-qPCR data for MALAT1 expression were analyzed by one-way ANOVA and Tukey post hoc test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n = 4 or 8 independent experiments as indicated.

Figure 6

uORF-targeting steric block ASOs do not activate a RNASEH1 5′ UTR-driven luciferase reporter construct (A) Schematic of experimental design for sequential plasmid and ASO transfection. Cells were first transfected with plasmids encoding RNASEH1 5′ UTR DLR constructs. After 24 h, cells were transfected with ASOs as indicated. Cells were subsequently harvested after a further 24 h. Renilla luciferase activity was determined for RNASEH1 5′ UTR constructs and signal normalized to firefly luciferase (encoded from a cistronically independent transgene cassette) for (B) HeLa cells (n = 5 independent experiments), and (C) HEK293T cells (n = 3 independent experiments). A gapmer targeting RLuc (100 nM) was used as a positive control for transfection. A TTG mutant in which the RNASEH1 uORF start codon is disrupted indicated the theoretical maximum of reporter upregulation. Values are mean + SD. Statistical significance was assessed by one-way ANOVA and Tukey post hoc test, ns, not significant; ∗p < 0.05. Statistical comparisons are to the RNASEH1 WT group unless otherwise indicated.