Aug 2026· Proceedings of the National Academy of Sciences of the United States of America· Vol 123 34, pp.
e2610782123
· 0 citations· 82 references
Medicine
TL;DR
A single uORF in KCNQ2 is identified that is highly repressive of protein translation and it is demonstrated that mutations disabling the uORF start codon enhance synthesis of encoded potassium channels and weaken ribosome engagement at the uORF.
Abstract
Upstream open reading frames (uORFs) within the 5'-untranslated region (5'-UTR) of messenger RNA transcripts can regulate protein translation. Despite widespread prevalence within the human genome, they remain unidentified for many clinically relevant genes. A gene frequently associated with neonatal-onset epilepsy is KCNQ2, which encodes a neuronal voltage-gated potassium channel subunit that functions to dampen neuronal excitability. Heterozygous loss-of-function pathogenic KCNQ2 variants are known to cause a range of neurodevelopmental disorders and epileptic encephalopathies, but there remains an unmet clinical need for patients harboring these variants. We identified a single uORF in KCNQ2 that is highly repressive of protein translation and demonstrated that mutations disabling the uORF start codon enhance synthesis of encoded potassium channels. Additionally, we show that adenine base editing of the uORF start codon can weaken ribosome engagement at the uORF and enhance translation of the protein in a neuron-like cell line. This study establishes a previously underexplored regulatory feature for KCNQ2 and highlights the importance of understanding uORFs for clinically relevant genes, both for assessing disease risk and therapeutic potential.
Neural activity-dependent translation is essential for synaptic plasticity and diverse brain functions. Translation involves not only canonical main open reading frames (mORFs) but also upstream ORFs (uORFs), which may regulate mORF expression. However, due to technical limitations, systematic investigation of activity-dependent uORFs and mORFs in brain tissues remains challenging. Here, we developed a ribosome tagging and purification strategy that bypasses the prolonged turnover of ribosomal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation. Applying this strategy to mouse hippocampal slices undergoing long-term potentiation, we identify hundreds of activity-induced mORFs and uORFs, including a previously unknown uORF from Egr1. We demonstrate that this Egr1-uORF translation is tightly regulated by neuronal activity, and its encoded peptide interacts with peroxisomal machinery, suggesting a potential link between synaptic stimulus and peroxisome biology. This study provides a useful technique and resources for deciphering molecular mechanisms underlying activity- and translation-dependent brain functions in health and disease. Investigation of activity-dependent protein synthesis selectively in activated neurons has long been a challenge. Here, the authors developed a technique to avoid the translational background from inactive neurons and uncovered previously unknown activity-dependent alternative translation with functional implication, such as the uORF of Egr1.
Nayan Suryawanshi, Hitoshi Uchida, R. Endo et al.· Nature Communications· 0 citations
CELF4 (CUGBP Elav-like family member 4), encoded by the human chromosome 18q12.2 locus, is an RNA-binding protein that recognizes UG-rich sequences within the 3′untranslated region (3′UTR) of target mRNAs to regulate splicing, stability, and local translation at the post-transcriptional level. Under physiological conditions, CELF4 exerts translational repression during synaptic development in the central nervous system (CNS), maintains excitatory homeostasis, and sets peripheral sensory thresholds; in cardiac fibroblasts, it is expressed at low levels and restricts baseline TGF-β signaling. In pathological states, CELF4 exhibits context-dependent bidirectional modulation: in autism spectrum disorder (ASD), major depressive disorder (MDD), epilepsy, chronic pain, and endometrial cancer, its downregulation or epigenetic silencing causes translational derepression of target mRNAs; in cardiac fibrosis, TGF-β1-induced upregulation suppresses FMO2 translation and activates the Smad2/3 pathway. Additionally, pleiotropic genetic loci near CELF4 have been linked to gut-brain axis comorbidities and obesity-hypertension syndromes. Clinically, CELF4 promoter methylation testing has entered validation trials for non-invasive endometrial cancer screening, and its haploinsufficiency has been incorporated into the genetic diagnosis of 18q12.2 microdeletion syndrome; pharmacological and gene-replacement strategies targeting CELF4 remain at the preclinical proof-of-concept stage. Here, we review the molecular regulatory networks of CELF4 and its mechanisms across multisystem diseases, discuss the current status and limitations of clinical translation, and may guide future research on diagnostic biomarkers and therapeutic strategies targeting this protein.
Qingsong Wang, Wenlong Yue, D. Lin et al.· Frontiers in Molecular Biosc...· 0 citations
Upstream open reading frames (uORFs) are widespread cis-regulatory elements that modulate translation initiation of downstream main ORFs (mORFs). Among them, overlapping uORFs (ouORFs) that overlap with mORFs are predicted to exert the strongest translational repression, yet they remain largely unexplored because of the difficulty of their identification. Here, we developed complementary computational approaches to systematically identify translated ouORFs from super-resolution ribosome profiling data in Arabidopsis. We identified 965 translated ouORFs alongside 7,180 canonical non-overlapping uORFs (nuORFs). We found that ouORFs exert substantially stronger translational repression than nuORFs, and that this repression depends primarily on Kozak context rather than uORF length. In addition, genes containing ouORFs or nuORFs have weaker mORF Kozak contexts than genes without uORFs, which may further reduce mORF translation. Moreover, ouORF translation promotes initiation downstream of the annotated mORF start codon, generating N-terminally truncated protein isoforms with altered domain composition and subcellular localization. Using ATPS2 as an example, we demonstrate that ouORF translation regulates alternative translation initiation to control the balance between chloroplast and cytosolic protein isoforms. Together, our findings establish ouORFs as a versatile class of translational regulatory elements that coordinate both protein abundance and protein diversity, providing the first genome-wide characterization of translated ouORFs in plants.
H. Wu, Yu Cheng, Isaiah D. Kaufman et al.· bioRxiv· 0 citations
The precise regulation of protein synthesis is essential for cellular function and survival. In particular, in neurons, dysregulated mRNA translation is linked to impaired memory formation and is a hallmark of neurodegenerative diseases. Neurons are characterized by tissue-specific, long 3′ untranslated regions (3′UTRs); in this study, we demonstrate that mRNA isoforms with these neuronal 3′UTRs are less efficiently translated than their short counterparts in Drosophila and mammalian brains. 3′UTR-dependent translation is based on a negative feedback mechanism centered around the two neural-enriched proteins ELAV and Pumilio. The long elav 3′UTR inhibits production of the neuronal ELAV protein, which in turn mediates 3′UTR extension of hundreds of neuronal genes. Those long 3′UTR isoforms are preferentially bound and translationally inhibited by Pumilio. The regulatory loop maintains optimal neuronal 3′UTR and protein levels in conditions of genetic and environmental perturbations; its disruption reduces animal viability and lowers stress resilience, and causes severe developmental phenotypes in flies and in human brain organoids. We propose 3′UTR-mediated translational control as an evolutionarily conserved mechanism for the maintenance of cell-type-specific proteostasis.
Sakshi Gorey, Hasan Can Ozbulut, Judit Carrasco et al.· bioRxiv· 0 citations
Regulation of gene expression in cells is mediated by RNA-binding proteins (RBPs), which act as adaptors connecting messenger RNA (mRNA) to enzymatic and structural components to achieve a distinct functional outcome. RBPs are enriched in intrinsically disordered regions (IDRs). These regions mediate multivalent interactions that lead to the expansion of a physical and functional network in cells and, therefore, play a pivotal role in mRNA processing. In this review, we highlight the role of IDRs in eukaryotic mRNA decay. IDRs drive the assembly of transient mRNA-protein complexes essential for mRNA degradation and regulate the catalytic activities of enzymes involved therein. Beyond these functions, IDRs connect different pathways of targeted mRNA decay, building a global functional network that dictates gene expression.
Sarah Lewandowski, L. Pommerening, Sutapa Chakrabarti· TIBS -Trends in Biochemical...· 0 citations
Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA decay pathway triggered by premature termination codons (PTCs). Although NMD is known to eliminate aberrant transcripts, how PTC position influences cell-to-cell heterogeneity in NMD and the resulting protein outputs remains unclear. Here, we used a single-cell NMD analysis system that quantifies cellular variability based on the GFP/mCherry fluorescence ratio. By combining this system with fluorescence-activated cell sorting (FACS), we show that PTC location critically determines not only NMD efficiency and its variability across cells, but also the spectrum of resulting protein products. These include truncated proteins arising from premature termination, full-length proteins generated through translational readthrough, and N-terminally truncated isoforms produced by downstream reinitiation. Our findings reveal that positional and cellular heterogeneity in NMD contribute to proteomic diversity and may underlie the variable phenotypic severity of genetic diseases caused by PTCs. This work establishes a framework for dissecting NMD regulation and its translational consequences. Highlights Protein-level readouts reflect mechanisms underlying NMD escape
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026
