bims-micpro 2025-07-20 papers

bims-micpro

Biomed News

on Discovery and characterization of microproteins

Issue of 2025–07–20
eight papers selected by
Thomas Farid Martínez, University of California, Irvine

Targeted Inhibition of Translation Initiation via 2'O-Methylation of Start Codons.
An expanded reference catalog of translated open reading frames for biomedical research.
Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii reveals untranslated small RNAs with ribosome occupancy.
A putative LncRNA RBM26-AS1-encoded micropeptide promotes colon cancer progression.
Non-canonical activation of MAPK signaling by the lncRNA ASH1L-AS1-encoded microprotein APPLE through inhibition of PP1/PP2A-mediated ERK1/2 dephosphorylation in hepatocellular carcinoma.
A LINC00472-encoded polypeptide impedes migration and proliferation through modulation of the HDAC2/SP1 axis in non-small cell lung cancer cells.
Long noncoding RNA FGD5-AS1-encoded micropeptides improve right heart functions of pulmonary arterial hypertension through restricting the synthesis of hyaluronic acid.
Peptide inhibitors: Breaking cancer code.

bioRxiv. 2025 Jun 17. pii: 2025.06.14.659565. [Epub ahead of print]

Targeted Inhibition of Translation Initiation via 2'O-Methylation of Start Codons.

Emmely A Patrasso, Hannah Serio, Adam Suh, Stephanie Mou, Kevin Vasquez, Siqi Wu, Yang Yi, Daniel Arango.

While protein synthesis typically initiates at an optimal AUG start codon, the 5' untranslated region (5'UTR) of mRNAs harbors non-canonical start codons that result in the translation of upstream Open Reading Frames (uORFs). However, the mechanisms underlying the selection of non- canonical start codons remain poorly understood. Structural analysis of translation pre-initiation complexes showed that the 2'OH group of the first nucleotide within start codons is monitored by 18S rRNA, allowing optimal translation initiation. We identified a chemical modification of mRNA, 2'O-methylation (N m ), that represses translation initiation by interfering with the interaction of mRNA and 18S rRNA, thereby preventing the pre-initiation complex from recognizing start codons. Significantly, we uncovered the presence of 2'O-methylation in the first nucleotide of non- canonical initiation sites within 5'UTRs, where it repressed upstream translation initiation. These findings revealed a potent regulatory function of 2'O-methylation in translation initiation and uORF expression, providing new insights into the mechanisms of non-canonical start codon selection in human cells.

DOI: https://doi.org/10.1101/2025.06.14.659565
bioRxiv. 2025 Jul 07. pii: 2025.07.03.662928. [Epub ahead of print]

An expanded reference catalog of translated open reading frames for biomedical research.

Sonia Chothani, Jorge Ruiz-Orera, Jack A S Tierney, Jim Clauwaert, Eric W Deutsch, M Mar Alba, Julie L Aspden, Pavel V Baranov, Ariel Alejandro Bazzini, Elspeth A Bruford, Marie A Brunet, Tristan Cardon, Anne-Ruxandra Carvunis, Claudio Casola, Jyoti Sharma Choudhary, Kellie Dean, Pouya Faridi, Ivo Fierro-Monti, Isabelle Fournier, Adam Frankish, Mark Gerstein, Norbert Hubner, Yunzhe Jiang, Manolis Kellis, Leron W Kok, Thomas F Martinez, Gerben Menschaert, Pengyu Ni, Sandra Orchard, Xavier Roucou, Joel Rozowsky, Michel Salzet, Mauro Siragusa, Sarah Slavoff, Michal I Swirski, Eivind Valen, Juan Antonio Vizcaino, Aaron Wacholder, Wei Wu, Zhi Xie, Yucheng T Yang, Robert L Moritz, Jonathan Mudge, Sebastiaan van Heesch, John R Prensner, Owen Jl Rackham.

Non-canonical (i.e., unannotated) open reading frames (ncORFs) have until recently been omitted from reference genome annotations, despite evidence of their translation, limiting their incorporation into biomedical research. To address this, in 2022, we initiated the TransCODE consortium and built the first community-driven consensus catalog of human ncORFs, which was openly distributed to the research community via Ensembl-GENCODE. While this catalog represented a starting point for reference ncORF annotation, major technical and scientific issues remained. In particular, this initial catalogue had no standardized framework to judge the evidence of translation for individual ncORFs. Here, we present an expanded and refined catalog of the human reference annotation of ncORFs. By incorporating more datasets and by lifting constraints on ORF length and start-codon, we define a comprehensive set of 28,359 ncORFs that is nearly four times the size of the previous catalog. Furthermore, to aid users who wish to work with ncORFs with the strongest and most reproducible signals of translation, we utilized a data-driven framework (i.e. translation signature scores) to assess the accumulated evidence for any individual ncORF. Using this approach, we derive a subset of 7,888 ncORFs with translation evidence on par with canonical protein-coding genes, which we refer to as the Primary set. This set can serve as a reliable reference for downstream analyses and validation, with a particular emphasis on high quality. Overall, this update reflects continual community-driven efforts to make ncORFs accessible and actionable to the broader research public and further iterations of the catalog will continue to expand and refine this resource.

DOI: https://doi.org/10.1101/2025.07.03.662928
bioRxiv. 2025 Jul 13. pii: 2025.04.08.647799. [Epub ahead of print]

Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii reveals untranslated small RNAs with ribosome occupancy.

Emma Dallon, Haley M Moran, Sadhana R Chidambaran, Arman Kian, Betty Y H Huang, Stephen D Fried, Jocelyne DiRuggiero.

Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized, however little is known about the impacts of oxidative stress at the translation level. Using the model archaeon Haloferax volcanii , we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded in these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins in H. volcanii , revealed additional regulatory layers and underscore the multifaceted architecture of stress-responsive regulatory networks.
Importance: Archaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. Using Haloferax volcanii as a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation.

DOI: https://doi.org/10.1101/2025.04.08.647799
Int J Surg. 2025 Jul 09.

A putative LncRNA RBM26-AS1-encoded micropeptide promotes colon cancer progression.

Qi Wu, Zhenling Ji.

   BACKGROUND: The functional diversity and mechanistic complexity of long non-coding RNAs (lncRNAs) exert various regulatory roles in cancer, and they have traditionally been annotated as non-coding genes. Currently, the coding potential of lncRNAs is gradually being revealed, however, their validation and mechanisms of action in cancer remain largely unknown.
METHODS: The expression, prognosis, and function of RBM26-AS1 in colon cancer were analyzed by bioinformatics, and its coding potential was predicted. The expression and localization of the RBM26-AS1 peptide were verified by constructing various fusion constructs, western blotting, and immunofluorescence. Cell proliferation and clonal formation experiments verified the functional effects of micropeptide on colon cancer cells. Co-immunoprecipitation (co-IP), mass spectrometry, and bioinformatic analysis were used to determine the possible mechanism of action of the micropeptide.
RESULTS: The lncRNA RBM26-AS1 could encode two micropeptides with different relative molecular masses. The RBM26-AS1 peptide promotes colon cancer cell growth and colony formation. Mechanistically, the RBM26-AS1 peptide may play a role in enhancing nucleocytoplasmic transport and protein processing in the endoplasmic reticulum, which may be different from the function of lncRNA itself. The inhibition of the RBM26-AS1 peptide impairs the proliferation ability of colon cancer cells.
CONCLUSIONS: A previously unknown micropeptide hidden in lncRNA RBM26-AS1 may contribute to colon cancer progression by enhancing nucleoplasmic transport and protein processing in the endoplasmic reticulum.

Keywords:  colon cancer; long non-coding rNAs (lncRNAs); micropeptide; tumour progression

DOI:  https://doi.org/10.1097/JS9.0000000000002834
J Exp Clin Cancer Res. 2025 Jul 11. 44(1): 200

Non-canonical activation of MAPK signaling by the lncRNA ASH1L-AS1-encoded microprotein APPLE through inhibition of PP1/PP2A-mediated ERK1/2 dephosphorylation in hepatocellular carcinoma.

Lei Zhao, Ke Si, Shenjian Luo, Lantian Zhang, Shuai Mao, Wenliang Zhang.

   BACKGROUND: MAPK/ERK1/2 signaling is often activated in hepatocellular carcinoma (HCC), yet classical RAS-RAF-MEK mutations are rare, indicating the involvement of non-canonical regulatory mechanisms. Long non-coding RNAs (lncRNAs) can encode microproteins that play key roles in cancer. LncRNA ASH1L-AS1 has coding potential, but its role in HCC remains unclear. Clarifying its role in MAPK signaling may uncover novel therapeutic targets for HCC.
METHODS: Translatable lncRNAs associated with HCC were identified by integrating data from the TCGA-LIHC cohort and the TransLnc database. The functional role of ASH1L-AS1 and its encoded microprotein APPLE was explored through in vitro and in vivo assays, such as CCK-8, EdU incorporation, wound healing, Transwell migration and invasion, and xenograft tumor models. Mechanistic investigations were conducted to elucidate molecular mechanisms and identify potential therapeutic strategies, including co-immunoprecipitation, mass spectrometry, ChIP-qPCR, luciferase reporter assays, truncation mutation analysis, immunofluorescence, Western blot, RNA sequencing, drug sensitivity analysis etc. RESULTS: A total of 696 translatable lncRNAs associated with HCC were identified, with their encoded products exhibiting specific subcellular localization. Among them, ASH1L-AS1 stood out due to strong translational evidence and its significant association with disease progression, poor prognosis, immunosuppressive tumor microenvironment, and estrogen signaling. We confirmed that ASH1L-AS1 encodes a microprotein, APPLE, which is stably expressed in HCC cells and consistently upregulated in tumor tissues regardless of RAS mutation status. Functionally, APPLE promotes ERK1/2 phosphorylation, activates MAPK signaling, and enhances HCC cell proliferation, migration, invasion, and tumor growth-effects reversed by APPLE knockdown or ERK1/2 inhibition. Mechanistically, APPLE binds to ERK1/2 and phosphatases PP1/PP2A, preventing ERK1/2 dephosphorylation and sustaining MAPK pathway activation. Additionally, the transcription factor E2F1 directly binds to the ASH1L-AS1 promoter (- 300 to - 290 bp), upregulating APPLE expression and further amplifying ERK1/2 signaling. Drug sensitivity analysis identified 220 treatment combinations potentially effective against HCC subtypes driven by hyperactivation of the E2F1-ASH1L-AS1/APPLE-ERK1/2 axis.
CONCLUSIONS: This study characterized APPLE as a novel oncogenic microprotein encoded by lncRNA ASH1L-AS1, uncovering a non-canonical mechanism of MAPK activation in HCC. The identified E2F1-ASH1L-AS1/APPLE-ERK1/2 signaling axis provides new insights into HCC pathogenesis and represents a promising target for precision therapy, though further validation in clinical cohorts and preclinical studies is needed.

Keywords:   ASH1L-AS1 ; Liver cancer; LncRNA encoded-microprotein; MAPK signaling; PP1/PP2A

DOI:  https://doi.org/10.1186/s13046-025-03465-w
Cancer Cell Int. 2025 Jul 16. 25(1): 263

A LINC00472-encoded polypeptide impedes migration and proliferation through modulation of the HDAC2/SP1 axis in non-small cell lung cancer cells.

Lei Xu, Haoyong Kuang, Haodong Peng, Sen Wu, Yu Bai, Xiangbo Jia, Wenjian Yao.

   OBJECTIVE: While long non-coding RNAs (lncRNAs) are increasingly recognized as sources of functional micropeptides, their roles in non-small cell lung cancer (NSCLC) remain poorly characterized. This study investigates the therapeutic potential and molecular mechanism of LINC00472-encoded polypeptide in NSCLC.
METHODS: Through integration of ribosome profiling, transcriptomics, and co-expression analysis, we systematically identified lncRNA-encoded polypeptides in NSCLC. Translational competence was validated via ribosome affinity purification (TRAP), Western blot, and immunofluorescence (IF). Functional assays (CCK-8, EdU, wound healing, transwell) and xenograft models assessed anti-tumor effects. HDAC2/SP1 interaction dynamics were analyzed by co-IP and luciferase reporter systems.
RESULTS: Multi-omics screening identified LINC00472 as a bifunctional transcript encoding a 15-aa polypeptide (LINC00472-ORF). LINC00472-ORF exhibited potent tumor-suppressive activity, reducing NSCLC proliferation and motility in vitro, while suppressing xenograft growth in vivo. Mechanistically, LINC00472-ORF disrupted HDAC2/SP1 interaction, inducing SP1 hyperacetylation, cytoplasmic retention, and transcriptional inactivation of downstream oncogenic genes.
CONCLUSION: We unveil LINC00472-ORF as a dual-function therapeutic agent that targets the HDAC2/SP1 axis to inhibit NSCLC progression.

Keywords:  Epigenetic-transcriptional regulation; HDAC2/SP1 axis; LINC00472; Multi-omics screening; NSCLC; lncRNA-encoded polypeptide

DOI:  https://doi.org/10.1186/s12935-025-03901-z
Int J Biol Macromol. 2025 Jul 14. pii: S0141-8130(25)06575-4. [Epub ahead of print] 146018

Long noncoding RNA FGD5-AS1-encoded micropeptides improve right heart functions of pulmonary arterial hypertension through restricting the synthesis of hyaluronic acid.

Yijun Chen, Qianhui Zhang, Xi Chen, Yahe Xu, Jing Guo, Jiajia Meng, Mingjie Zhang, Bo Wang, Zhuoming Xu.

  Pulmonary arterial hypertension (PAH) is marked by elevated vascular resistance, right ventricular (RV) failure, and poor clinical outcomes. Current therapies primarily target pulmonary vascular hemodynamics, necessitating novel strategies to address RV remodeling. In this study, we investigated the role of lncRNA FGD5-AS1 and its micropeptides in PAH and RV remodeling, focusing on their effects on hyaluronic acid (HA) synthesis and extracellular matrix organization. FGD5-AS1 expression was significantly reduced in peripheral blood mononuclear cells of PAH patients and inversely correlated with HA levels and disease severity. Functional studies using FGD5-AS1 knockout (KO) in the AC16 human cardiomyocyte cell line led to upregulation of HAS2, increased HA production and activation of TLR4, contributing to pro-fibrotic and pro-hypertrophic responses. In a monocrotaline-induced PAH rat model, overexpression of FGD5-AS1 encoded micropeptide Pep1 reduced HA synthesis, suppressed heart failure biomarkers (NPPA, NPPB), and improved cardiac function, while Pep2 showed limited benefits. These findings demonstrate that FGD5-AS1 exerts protective effects in PAH by modulating HA synthesis through HAS2 regulation. The micropeptides, particularly Pep1, offer promising therapeutic potential for improving RV function and remodeling in PAH. This study highlights FGD5-AS1 and its derived micropeptides as novel therapeutic targets for PAH, providing new strategies to address RV dysfunction in this debilitating disease.

Keywords:  FGD5-AS1; HAS2; Hyaluronic acid; Pulmonary arterial hypertension

DOI:  https://doi.org/10.1016/j.ijbiomac.2025.146018
Eur J Med Chem. 2025 Jul 11. pii: S0223-5234(25)00726-3. [Epub ahead of print]297 117961

Peptide inhibitors: Breaking cancer code.

Fleur Coburn, Yanyamba Nsereko, Amy Armstrong, Othman Al Musaimi.

  Tumour signalling pathways play a pivotal role in tumorigenesis by controlling key cellular processes, including growth, proliferation, metastasis, and survival. Conventional treatments like chemotherapy often face limitations such as non-specific toxicity and drug resistance. Peptide inhibitors have gained attention as a promising therapeutic alternative due to their high selectivity in disrupting oncogenic pathways via protein-protein interactions (PPIs) and ligand binding. Currently, two out of 31 peptide-based cancer drugs have demonstrated signalling inhibition, with several others under investigation. However, challenges related to stability, delivery, and resistance persist, prompting innovations in peptide design, such as cyclisation and nanoparticle-based delivery systems. This review examines strategies to enhance peptide drug efficacy, explores the mechanisms by which peptide inhibitors target key pathways like the rat sarcoma protein (RAS) and mammalian target of rapamycin (mTOR) pathways, and highlights ongoing research on peptide-based interventions. Key examples include RAS-targeting peptides such as KRpep-2D, cyclo-CRVLIR, L5UR, RAS-binding peptide (RBP), the mutant KRAS peptide vaccine combined with Nivolumab and Ipilimumab, cyclorasin B4-27, and LUNA18, as well as mTOR-targeting peptides like P1_WT, PDHK1-241aa, TRIM1-269aa, and the micropeptide human small regulatory polypeptide of amino acid response (hSPAR). Among these, the mutant KRAS peptide vaccine (with Nivolumab and Ipilimumab) and LUNA18 demonstrate promising clinical potential and are currently undergoing trials.

Keywords:  Cancer; Inhibitors; Peptides; RAS; Signalling pathways; mTOR

DOI:  https://doi.org/10.1016/j.ejmech.2025.117961