bims-micpro 2025-06-08 papers

Elife. 2025 Jun 06. pii: RP104074. [Epub ahead of print]14

Upstream open reading frames buffer translational variability during Drosophila evolution and development.

Yuanqiang Sun, Yuange Duan, Peixiang Gao, Chenlu Liu, Kaichun Jin, Shengqian Dou, Wenxiong Tang, Hong Zhang, Jian Lu.

Protein abundance tends to be more evolutionarily conserved than mRNA levels both within and between species, yet the mechanisms underlying this phenomenon remain largely unknown. Upstream open reading frames (uORFs) are widespread cis-regulatory elements in eukaryotic genomes that regulate translation, but it remains unclear whether and how uORFs contribute to stabilizing protein levels. In this study, we performed ribosome translation simulations on mRNA to quantitatively assess the extent to which uORF translation influences the translational variability of downstream coding sequences (CDSs) across varying contexts. Our simulations revealed that uORF translation dampens CDS translational variability, with buffering capacity increasing in proportion to uORF translation efficiency, length, and number. We then compared the translatomes at different developmental stages of two Drosophila species, demonstrating that uORFs buffer mRNA translation fluctuations during both evolution and development. Experimentally, deleting a uORF in the bicoid (bcd) gene-a prominent example of translational buffering-resulted in extensive changes in gene expression and phenotypes in Drosophila melanogaster. Additionally, we observed uORF-mediated buffering between primates and within human populations. Together, our results reveal a novel regulatory mechanism by which uORFs stabilize gene translation during development and across evolutionary time.

Keywords: TASEP modeling; bicoid; drosophila melanogaster; drosophila simulans; evolutionary biology; genetics; genomics; humans; translation evolution; translational regulation; uORF

DOI: https://doi.org/10.7554/eLife.104074

bioRxiv. 2025 May 18. pii: 2025.05.18.654700. [Epub ahead of print]

Upstream open reading frame inactivation augments GATA4 translation and cardiomyocyte hypertrophy in mice.

Omar M Hedaya, Feng Jiang, Uday Baliga, Aleksandr Ivanov, Si Chen, Jack L Schwartz, Yui Kawakami, Chen Yan, Peng Yao.

Upstream open reading frames (uORFs) are short peptide-encoding sequences located in the 5' untranslated region (5' UTR) of mRNAs, enabling translational repression of main (m)ORFs. While uORFs are found in ∼50% of mRNAs in humans, our understanding of their biological function remains limited. This study aims to elucidate the role of the uORF in the 5' UTR of the Gata4 (GATA binding protein 4) gene in cardiac biology by inactivating its start codon (ΔuORF) in the mouse genome. Our investigation reveals that mice with Gata4 uORF inactivation manifest spontaneous cardiac hypertrophy without apparent fibrosis as they age. Utilizing single-nucleus RNA sequencing (snRNA-seq), we uncovered significant transcriptional variations between wild-type (WT) and ΔORF mice. Notably, mRNAs associated with sarcomeres and contractile functions show heightened expression levels, reflecting the hypertrophic phenotype. Notably, at least nine upregulated genes are GATA4-bound targets in mouse ventricles. Functional assessments of isolated primary adult cardiomyocytes confirmed enhanced hypertrophy and contractility in ΔORF mice. Additionally, we employed single-nucleus transposase-accessible chromatin (snATAC)-seq to investigate changes in chromatin accessibility. Our results indicated increased accessibility within specific transcription-regulatory elements linked to elevated gene transcription. These putative cis-regulatory elements (pCREs) are significantly enriched in MEF2 (myocyte enhancer factor 2) binding motifs. In vitro luciferase reporter assays further supported the regulatory potential of three of these pCREs, highlighting their role in the transcriptional enhancement of three GATA4 target genes bound by MEF2 and GATA4. These findings illuminate the role of uORF in negatively regulating GATA4 protein expression and cardiomyocyte hypertrophy at the organismal level and provide a novel therapeutic target for cardiac pathogenesis.

DOI: https://doi.org/10.1101/2025.05.18.654700

Trends Microbiol. 2025 May 30. pii: S0966-842X(25)00150-7. [Epub ahead of print]

New genes on the block: additional open reading frames in the genomes of geminiviruses.

Huang Tan, Fangfang Li, Xueping Zhou, Rosa Lozano-Durán.

In their DNA genomes, the devastating plant-infecting geminiviruses contain partially overlapping genes which encode multifunctional proteins. For decades, geminiviral genomes were believed to contain four to eight open-reading frames (ORFs); now, a burst of independent works has found evidence for the existence of additional ORFs, revealing that their coding space remains to be fully explored.

Keywords: coding space; geminiviruses; gene; ribosome profiling; small open reading frames; virulence factor

DOI: https://doi.org/10.1016/j.tim.2025.05.003

Proc Natl Acad Sci U S A. 2025 Jun 10. 122(23): e2502155122

Control of seed-to-seedling transition by an upstream open reading frame in ABSCISIC ACID DEFICIENT2.

Zhen Wang, Xiaofan Zhang, Can Zhou, Xiaofeng Cao.

The start of seed germination is a major decision point in plant life cycle, which relies on seed stored mRNA. However, the underlying translational mechanism remains less illustrated. Here, we demonstrate that inhibiting translation using translation inhibitors and ribosome-defective mutants delays germination in Arabidopsis. Through comprehensive transcriptome deep sequencing (RNA-seq) and polysome profiling analyses, we elucidated the dynamic interplay of regulation at the transcriptional and translational levels during germination. We show that delayed germination in some ribosome-defective mutants is partially regulated by the gene ABSCISIC ACID DEFICIENT2 (ABA2), with an upstream open reading frame (uORF) in the 5' untranslated region that represses translation of the downstream ORF encoding ABA2. In addition, disrupting rice OsABA2 uORF inhibited preharvest sprouting (PHS). Furthermore, we found two main haplotypes for the uORF among rice cultivars that result in different OsABA2 expression levels, thus contributing to diverse PHS phenotypes. This work highlights the critical role of translational control and genetic variation in seed dormancy and germination, with implications for crop improvement.

Keywords: ABA; preharvest sprouting; seed dormancy and germination; translation; uORF

DOI: https://doi.org/10.1073/pnas.2502155122

Cancer Immunol Res. 2025 Jun 03.

CTG-initiated cryptic peptide translation up and downstream of a canonical ATG start codon is enhanced by TLR stimulation and induces tumor regression in mice.

Ziye Song, Youkyung Lim, Anneloes van Krimpen, Mitchell A A Geleijnse, Manon Messchendorp, Jane S A Voerman, Ling Li, Emma G M Tondeur, Gunja Mishra, Brett J Hos, Dwin G B Grashof, Ralph Stadhouders, Harmen J G van de Werken, Peter D Katsikis, Christopher Schliehe.

Cytotoxic T-lymphocytes (CTLs) screen cells for signs of infection and transformation by recognizing peptides displayed on major histocompatibility complex (MHC) class I molecules. Next to canonical ATG-initiated open reading frames (ORFs), non-canonical translation can result in synthesis of non-conventional or `cryptic´ polypeptides. These can originate from translation initiation at non-canonical start codons, a process previously associated with inflammation and oncogenic transformation. Cryptic translation products are efficiently presented on MHC class I molecules and therefore increasingly recognized as potential targets for cancer immunotherapy. Here, we studied how localization of a CTG-initiated ORF relative to a canonical ATG start codon can influence cryptic expression after innate immune stimulation. We generated immortalized C57BL/6J mouse-derived bone marrow progenitor cells (HoxB8) expressing tandem minigene constructs, which encoded a CTG-driven chicken ovalbumin-derived SIINFEKL (S8L) epitope (CTG-S8L; H-2Kb-restriced) either up or downstream of a canonical ATG-initiated UTY-derived peptide WI9 (ATG-WI9; H-2Db-restriced). Treatment of HoxB8-derived macrophages with Toll-like receptor agonists enhanced position-independent CTG-S8L translation, without affecting ATG-driven expression. Downstream CTG-S8L translation was driven by leaky scanning or ribosome re-initiation rather than read-through translation. Mouse AE17 mesothelioma and B16F10 melanoma cells expressing cryptic S8L either up or downstream of a canonical ORF were efficiently killed by H-2Kb/S8L-restriced OT-I T-cells in vitro, even though their antigen expression levels were extremely low. Mice implanted with tumors expressing cryptic S8L showed delayed tumor progression in vivo. In summary, our study contributes to the characterization of non-canonical start codon-driven cryptic antigen translation and highlights its potential for cancer immunotherapy.

DOI: https://doi.org/10.1158/2326-6066.CIR-24-0467

Proteomics. 2025 Jun 02. e00464

A Survey of Annotated smORF-Encoded Polypeptides in Bacteria Interacting With Eukaryotic Hosts.

Gonzalo Zapata, Ezequiel G Mogro, Abril L Pagnutti, Macarena Algañaras, Pilar Cañameras, Walter O Draghi, Antonio Lagares, Daniela Bottero, Mauricio J Lozano.

smORF encoded polypeptides (SEPs) are difficult to predict due to their small size. While modern genome annotation tools are capable of identifying smORFs, their reliability is often uncertain. Furthermore, experimental validation of smORFs has primarily focused on a limited set of model organisms. Here, we conduct a comprehensive analysis of annotated smORFs in a diverse range of bacteria interacting with eukaryotic hosts. Our analysis revealed that bacterial genomes typically harbor between 100 and 300 annotated smORFs, predominantly encoding SEPs exceeding 40 residues and annotated as hypothetical proteins. We show that functional annotation of SEPs can be improved to some extent with the currently available resources, and that SEPs exhibit distinct functional profiles in bacteria associated with different host types (plant vs. animal). We also found that most of the experimentally validated SEPs are conserved and that all the annotated SEPs begin with methionine, although that is not always the case for the experimentally validated ones. Our findings underscore the need for improved annotation methods and further experimental characterization to fully understand the functional roles and evolutionary significance of smORFs in bacteria-host interactions.

Keywords: SEP; micro peptides; prokaryote; sORF; smORF; small ORF

DOI: https://doi.org/10.1002/pmic.202400464