Application of antisense technology in the control of upstream open reading frame-mediated translation
Keywords: RNA editing
haploinsufficiency
translation
ribosome profiling
translational regulation
protein upregulation
oligonucleotides
uORF; Genetic disease; Genetic medicines

Upstream open reading frames (uORFs) are cis-regulatory sequence elements characterized by an initiation codon located in the 5′ untranslated region (5′ UTR) and an associated in-frame termination codon located upstream or downstream of the primary ORF (pORF). uORFs have been found to inhibit translational initiation at the pORF by sequestering ribosomes through premature initiation in the 5′ UTR. It was therefore hypothesized that that the disruption of uORFs would result in increased translation at the pORF.
Initially, we predicted occurrences of uORFs in the human transcriptome and generating a database (uORFeome) which annotates uORF features. To better understand the complex mechanisms by which uORFs inhibit protein expression, large-scale transcriptomics and proteomics datasets from 32 human tissues were combined with uORF predictions to perform a comprehensive bioinformatics analysis of the influence of uORF features. uORF Detective – a transcriptome-wide uORF visualization tool, was developed to enable the study of uORFs in context of ribosome profiling data. A deep learning model was used to predict uORF effects and discover novel sequence features with significant effects on uORF translational repression.
The model predicted that AUG-to-UUG uORF initiation codon changes would result in translational derepression (a well-established uORF property), and that minimal uORFs (an initiation codon immediately followed by a stop codon) would also be inhibitory (an unexpected finding). Furthermore, the presence of a tryptophan residue at position 5 of the uORF-encoded peptide was predicted to enhance uORF-mediated repression. All of these predictions were subsequently confirmed experimentally using reporter assays.
Additional bioinformatics and reporter assay analyses revealed a lack of activity of uORFs proximal to the cap, a discovery of initiation at a minimal uORF in the 5′ UTR of the ATF4 gene, identification of potentially therapeutically-relevant uORF target sites that may be utilized to upregulate expression of the BDNF and SCN1A genes, as well as position- and Kozak sequence-dependent effects on uORF activity. This work demonstrates that in cases where initiation at uORFs inhibits productive translation at the pORF, the disruption of the uORF initiation codon can result in increased translation of the pORF. Such interference with regulatory uORFs may be of benefit in multiple medical conditions where an increase in protein levels is desirable (e.g., haploinsufficiency disorders).
Oligonucleotide (ON) targeting strategies were explored to evaluate their ability to interfere with uORF activity. Steric block ONs were utilized to target uORF initiation codons. No upregulation was observed in any experiment using this strategy, suggesting that the previously published findings are not reproducible. As an alternative approach, RNA editing ONs were used to disrupt the uORF initiation codon by recruiting adenosine (A) deaminases acting on RNA (ADAR) enzymes. Sequence-specific RNA editing was observed in a variety of endogenous uORF targets at low efficiencies and in some cases, reporter gene activation was also observed. The importance of sequence context surrounding the target editing site was identified as a factor in successful editing, with C at the 5′ of the target A being particularly unfavorable. The inclusion of inosine (I) nucleotides opposite of C in the in the nearest neighbor position was found to partially recover RNA editing activity. While this editing was observed in context of interferon alpha (IFNα)-induced ADAR overexpression, future chemistry optimization may facilitate the generation of higher potency RNA editing ONs, which can be used to activate gene expression by interfering with uORFs.