Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

Gangras, P.; Gallagher, T. L.; Patton, R. D.; Yi, Z.; Parthun, M. A.; Tietz, K. T.; Deans, N. C.; Bundschuh, R.; Amacher, S. L.; Singh, G.

doi:10.1101/677666

bioRxiv · 10.1101/677666

Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

Gangras, P.·Gallagher, T. L.·Patton, R. D.·Yi, Z.·Parthun, M. A.·Tietz, K. T.·Deans, N. C.·Bundschuh, R.·Amacher, S. L.·Singh, G.

View original source ↗Export metadata ↓

Abstract

The Exon Junction Complex (EJC) regulates many steps in post-transcriptional gene expression and is essential for cellular function and organismal development; however, EJC-regulated genes and genetic pathways during development remain largely unknown. To study EJC function during zebrafish development, we first established that zebrafish EJCs mainly bind [~]24 nucleotides upstream of exon-exon junctions, and are also detected at more distant non-canonical positions. We then generated mutations in two zebrafish EJC core genes, rbm8a and magoh, and observed that homozygous mutant embryos show paralysis, muscle disorganization, neural cell death, and motor neuron outgrowth defects. Coinciding with developmental defects, mRNAs subjected to Nonsense-Mediated mRNA Decay (NMD) due to translation termination [≥] 50 nts upstream of the last exon-exon junction are upregulated in EJC mutant embryos. Surprisingly, several transcripts containing 3'UTR introns (3'UI) < 50 nts downstream of a stop codon are also upregulated in EJC mutant embryos. These proximal 3'UI-containing transcripts are also upregulated in NMD-compromised zebrafish embryos, cultured human cells, and mouse embryonic stem cells. Loss of function of foxo3b, one of the upregulated proximal 3'UI-containing genes, partially rescues EJC mutant motor neuron outgrowth. In addition to foxo3b, 166 other genes contain a proximal 3'UI in zebrafish, mouse and humans, and these genes are enriched in nervous system development and RNA binding functions. A proximal 3'UI-containing 3'UTR from one of these genes, HNRNPD, is sufficient to reduce steady state transcript levels when fused to a {beta}-globin reporter in HeLa cells. Overall, our work shows that genes with stop codon-proximal 3'UIs encode a new class of EJC-regulated NMD targets with critical roles during vertebrate development.

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Gangras, P., Gallagher, T. L., Patton, R. D., Yi, Z., Parthun, M. A., Tietz, K. T., Deans, N. C., Bundschuh, R., Amacher, S. L., Singh, G.. 2019-06-21. Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay. https://doi.org/10.1101/677666

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Hypoxic Stress Induces LARP1-Driven Stress Granules That Protect mRNAs from Decay to Sustain Mitotic Cell Cycle Progression

Hypoxia is a defining feature of solid tumours that elicits extensive transcriptional and post-transcriptional adaptations. Stress granules (SGs) are frequently observed under hypoxia, yet how they assemble and whether they contribute to hypoxic gene regulation remain poorly understood. Here, we show that hypoxia induces SGs through an assembly mechanism distinct from that of classical acute stresses. Hypoxia SG formation occurs independently of the integrated stress response and instead requires the HIF1-REDD1-mTORCC1-4EBP1 axis. Remarkably, hypoxia SGs assemble despite robust ongoing translation, suggesting that additional mechanisms lower the threshold for RNA condensation. We identify LARP1 as a potential determinant of this process, where it promotes G3BP-dependent SG assembly specifically during hypoxia. Despite their distinct assembly mechanism, hypoxia and arsenite SGs recruit broadly similar RNA populations, with selective differences validated by single-molecule FISH, suggesting that conserved principles govern RNA partitioning across different SG states. Functionally, sequestration into hypoxia SGs correlates with enhance protein expression whereby a subset of mRNAs are protected from decay. Transcriptomic analyses further reveal that this protection sustains a broader gene-expression program enriched for MCM complex and mitotic cell-cycle regulators. Accordingly, disruption of hypoxia SGs reduces mitotic progression and proliferative competence during prolonged hypoxia. Together, our findings establish LARP1-mediated condensation as a mechanism that enables SG assembly despite ongoing translation and reveal that SG sequestration preserves selected mRNAs from decay to sustain the gene-expression programs required for long-term adaptation to hypoxia.

molecular biology↗

bioRxiv2026

Age-associated repression of HNF4α is a driver and potential therapeutic target in liver metabolic dysfunction

Aging is a major risk factors for metabolic dysfunction-associated steatotic liver disease (MASLD). However, the mechanisms by which aging disrupts hepatocyte transcriptional networks to drive metabolic dysfunction remain poorly understood. Hepatocyte nuclear factor 4 alpha (HNF4) is a master regulator of hepatocyte identity and metabolic homeostasis, but its role in liver aging has not been defined. Here, we perform a comprehensive multi-omic profiling of young and aged mouse livers combined with targeted HNF4 functional studies to define how aging remodels HNF4-dependent gene regulation and whether HNF4 can be a target to reverse age-associated liver dysfunction. Despite preserved HNF4 expression with age, aging was associated with reduced HNF4 chromatin occupancy and transcriptional activity, accompanied by epigenetic remodeling, including changes in histone acetylation, chromatin accessibility, and DNA methylation. Integration and comparison of HNF4 CUT&RUN with hepatocyte-specific Hnf4a deletion in young and aged livers identified high-confidence overlapping but distinct young- and old- specific HNF4 target gene networks. In young hepatocytes, HNF4 coordinates two complementary transcriptional programs: activation of genes governing hepatocyte identity, lipid metabolism, and metabolic function, while simultaneously inhibiting alternative developmental, proliferative, and inflammatory programs. Spatial transcriptomic analyses further revealed zone-specific specialization of HNF4-dependent gene programs. Aging selectively disrupted these regulatory networks, resulting in compromised liver identity and metabolic gene programs, de-repression of inflammatory pathways and alternative cellular identity programs and disrupted hepatocyte zonation, consistent with age-dependent lipidomic and metabolic changes, hepatic fat accumulation, and immune infiltration. Hepatocyte-specific Hnf4a deletion in young mice recapitulated key features of liver aging, including impaired hepatocyte identity, metabolic dysfunction, steatosis, inflammatory activation and altered hepatic immune-cell composition, while deletion in aged mice further exacerbated steatosis, inflammation, and hepatic dysfunction. Conversely, hepatocyte-specific HNF4 restoration in aged mice significantly reduced hepatic steatosis and lipid accumulation, restored fatty acid metabolic programs, and partially reversed age-associated transcriptional changes. However, inflammatory and interferon-associated programs were not uniformly reversed, revealing differential responsiveness of metabolic and inflammatory pathways to HNF4 restoration. Together, these findings identify age-associated impairment of HNF4 activity as a functional contributor to liver aging, linking dysregulation of hepatocyte identity, metabolic homeostasis, and inflammatory control to age-associated hepatic dysfunction. Importantly, the selective reversibility of metabolic defects following HNF4 restoration establishes HNF4 as a promising strategy for preserving hepatocyte homeostasis and reversing age-associated hepatic metabolic dysfunction.

molecular biology↗

bioRxiv2026

A Human Tumor Virus Recruits HSP70 Chaperone to Remodel Its Replisome on DNA

Human Merkel cell polyomavirus (MCV) large T (LT) protein functions as a replication helicase analogous to the cellular MCM complex but requires a second MCV protein, small T (sT), for efficient LT-initiated replication. Both LT and sT encode highly-conserved DnaJ domains that bind to the cellular refolding molecule, heat shock protein 70 (HSP70). Using single-molecule imaging and FRET, we find that sT recruits dimeric HSP70 to serve as a molecular bridge between sT and LT on DNA and that this complex facilitates remodeling LT into a functional double-hexameric helicase. Disruption of LT-HSP70-HSP70-sT interaction with HSP70 inhibitors or by mutations to the LT-sT DnaJ domains collapse the LT-HSP70-HSP70-sT complex, abolishing sT-enhanced LT dodecamerization and ATP-dependent DNA unwinding. Assembly of virus-free mammalian MCM3, MCM4 and MCM6 in nuclear extracts similarly recruits HSP70 to DNA suggesting that Hsp70 remodeling is used for efficient eukaryotic assembly of the multi-protein replication machinery.

molecular biology↗