Biology subjects

Jad, Y.

Publications and source records attributed to Jad, Y..

3 recordsLinked to original sources

bioRxiv2025

Toward a model of uORF-mediated translational control: An integrated bioinformatic and experimental approach

Upstream open reading frames (uORFs) are short translated regions that occur in the 5{square} untranslated regions (5{square} UTRs) of mRNA transcripts where they primarily serve to repress expression translation of the downstream primary open reading frame (pORF). Their widespread presence across mammalian transcriptomes suggests an important role in shaping the proteome, although the mechanistic basis of their regulatory effects remain incompletely understood. Here we present an integrated experimental and computational investigation into the features that govern uORF-mediated translation control. Using high-resolution proteomics data from 29 healthy human tissues and machine learning-based simulations, we have systematically dissected how features including uORF length, amino acid composition, start codon position, stop codon position, and Kozak context influence repressive activity, and performed experimental validation using reporter gene constructs. We also investigated how multiple uORFs within a single 5{square} UTR can interact in synergistic or antagonistic ways, with the potential to produce counterintuitive effects on pORF translation. From these studies, we present a model of uORF function, suggesting a hierarchy of uORF feature importance, and proposing that a combination of uORF translation initiation probability, ribosome recycling rate, intercistronic ternary complex recharging requirements, and ribosome stalling mechanisms underlie uORF repressive activity. Together, these studies provide a comprehensive view of the molecular logic underlying uORF activity, offering new insights into their endogenous and highlighting their potential as targets for drug development.

biochemistry↗

bioRxiv2025

Dysregulated MEG3 in Myotonic Dystrophy 1: nuclear retention, pathological role, and therapeutic correction by antisense conjugates.

MEG3, a long non-coding RNA (lncRNA), has been shown to play a critical role in regulating apoptosis. Its downregulation inhibits apoptosis in cancer cells, whereas its upregulation has been associated with cell death in both cardiovascular disease and, more recently, Alzheimers Disease. Here we show that MEG3 is upregulated in Myotonic Dystrophy 1 (DM1). Specifically, we show MEG3 upregulation by several-fold in DM1 human muscle cells and in two DM1 mouse models, HSA-LR and LC15. In human DM1 muscle cells we observe nuclear retention of MEG3 and an increase in its transcript diversity. Furthermore, we observe a general trend of nuclear retention in DM1 affecting lncRNAs and microRNAs (miRNAs), in contrast to mRNAs, when compared to healthy cells. This altered nuclear retention may contribute to the pathological effects of non-coding RNA dysregulation in DM1. Importantly, we demonstrate that treatment with antisense conjugates targeting the repeat expansion causing DM1, an approach currently being tested in Clinical Trials, corrects MEG3 levels in HSA-LR mice, without additional therapeutic interventions targeting MEG3.

molecular biology↗

bioRxiv2024

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

Upstream open reading frames (uORFs) are cis-regulatory motifs that are predicted to occur in the 5' 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 the RNASEH1 uORF described in the study by Liang et 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 a MALAT1-targeting gapmer ASO as a positive control. We conclude that previously-described RNASEH1 uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.

biochemistry↗