bims-micpro 2025-02-23 papers

bims-micpro

Biomed News

on Discovery and characterization of microproteins

Issue of 2025–02–23
seven papers selected by
Thomas Farid Martínez, University of California, Irvine

Non-canonical ORFs-derived protein products in mitochondria: A multifaceted exploration of their functions in health and disease.
The hidden bacterial microproteome.
Unveiling conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling.
Short internal open reading frames repress the translation of N-terminally truncated proteoforms.
Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression.
NCBP2-AS2 is a mitochondrial microprotein, regulates energy metabolism and neurogenesis, and is downregulated in Alzheimer's disease.
The LINC01315-encoded small protein YAPer-ORF competes with PRP4k to hijack YAP signaling to aberrantly promote cell growth.

Protein Sci. 2025 Mar;34(3): e70053

Non-canonical ORFs-derived protein products in mitochondria: A multifaceted exploration of their functions in health and disease.

Ikram Ajala, Benoît Vanderperre.

  Traditionally, eukaryotic mRNAs were perceived as inherently monocistronic. However, recent insights from ribosome profiling (Ribo-seq) and proteomics studies challenge this paradigm. These investigations reveal that, beyond the currently annotated reference proteins (RefProts), there exist additional proteins known as alternative proteins (AltProts) and small open reading frames derived microproteins encoded in regions of mRNAs previously considered untranslated or in non-coding transcripts. This experimental evidence broadens the spectrum of functional proteins within cells, tissues, and organs, potentially offering crucial insights into biological processes. Notably, a significant proportion of these newly identified AltProts and microproteins demonstrates localization in mitochondria, contributing to the functions of mitochondrial complexes. This review delves into the overlooked realm of the alternative proteome within mitochondria, exploring the role of nuclear or mitochondrial-genome-encoded AltProts and microproteins in physiological and pathological cellular processes.

Keywords:  alternative open reading frames (AltORFs); alternative proteins (AltProts); mitochondria; proteome; small ORF (sORF)

DOI:  https://doi.org/10.1002/pro.70053
Mol Cell. 2025 Feb 11. pii: S1097-2765(25)00059-0. [Epub ahead of print]

The hidden bacterial microproteome.

Igor Fesenko, Harutyun Sahakyan, Rajat Dhyani, Svetlana A Shabalina, Gisela Storz, Eugene V Koonin.

  Microproteins encoded by small open reading frames comprise the "dark matter" of proteomes. Although microproteins have been detected in diverse organisms from all three domains of life, many more remain to be identified, and only a few have been functionally characterized. In this comprehensive study of intergenic small open reading frames (ismORFs, 15-70 codons) in 5,668 bacterial genomes of the family Enterobacteriaceae, we identify 67,297 clusters of ismORFs subject to purifying selection. Expression of tagged Escherichia coli microproteins is detected for 11 of the 16 tested, validating the predictions. Although the ismORFs mainly code for hydrophobic, potentially transmembrane, unstructured, or minimally structured microproteins, some globular folds, oligomeric structures, and possible interactions with proteins encoded by neighboring genes are predicted. Complete information on the predicted microprotein families, including evidence of transcription and translation, and structure predictions are available as an easily searchable resource for investigation of microprotein functions.

Keywords:  bacterial microproteins; evolution of microproteins; intergenic regions; protein structure prediction; smORFs; small open reading frames

DOI:  https://doi.org/10.1016/j.molcel.2025.01.025
Nat Commun. 2025 Feb 18. 16(1): 1707

Unveiling conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling.

Lisa Bertrand, Annika Nelde, Bertha Cecilia Ramirez, Isabelle Hatin, Hugo Arbes, Pauline François, Stéphane Demais, Emmanuel Labaronne, Didier Decimo, Laura Guiguettaz, Sylvie Grégoire, Anne Bet, Guillaume Beauclair, Antoine Gross, Maja C Ziegler, Mathias Pereira, Raphaël Jeger-Madiot, Yann Verdier, Joelle Vinh, Sylvain Cardinaud, Stéphanie Graff-Dubois, Audrey Esclatine, Cécile Gouttefangeas, Marcus Altfeld, Laurent Hocqueloux, Assia Samri, Brigitte Autran, Olivier Lambotte, Hans-Georg Rammensee, Emiliano P Ricci, Juliane Walz, Olivier Namy, Arnaud Moris.

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the translatome of HIV-1 in infected CD4+ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Reading Frames (sORFs) that are distributed across the HIV genome including the UTR regions. Using a database of HIV genomes, we observe that most ARF amino-acid sequences are likely conserved among clade B and C of HIV-1, with 8 ARF-encoded amino-acid sequences being more conserved than the overlapping CDSs. Using T cell-based assays and mass spectrometry-based immunopeptidomics, we demonstrate that ARFs encode viral polypeptides. In the blood of people living with HIV, ARF-derived peptides elicit potent poly-functional T cell responses mediated by both CD4+ and CD8+ T cells. Our discovery expands the list of conserved viral polypeptides that are targets for vaccination strategies and might reveal the existence of viral microproteins or pseudogenes.

DOI: https://doi.org/10.1038/s41467-025-56773-2
EMBO Rep. 2025 Feb 17.

Short internal open reading frames repress the translation of N-terminally truncated proteoforms.

Raphael Fettig, Zita Gonda, Niklas Walter, Paul Sallmann, Christiane Thanisch, Markus Winter, Susanne Bauer, Lei Zhang, Greta Linden, Margarethe Litfin, Marina Khamanaeva, Sarah Storm, Christina Münzing, Christelle Etard, Olivier Armant, Olalla Vázquez, Olivier Kassel.

  Internal translation initiation sites, as revealed by ribosome profiling experiments can potentially drive the translation of many N-terminally truncated proteoforms. We report that internal short open reading frame (sORF) within coding sequences regulate their translation. nTRIP6 represents a short nuclear proteoform of the cytoplasmic protein TRIP6. We have previously reported that nTRIP6 regulates the dynamics of skeletal muscle progenitor differentiation. Here we show that nTRIP6 is generated by translation initiation at an internal AUG after leaky scanning at the canonical TRIP6 AUG. The translation of nTRIP6 is repressed by an internal sORF immediately upstream of the nTRIP6 AUG. Consistent with this representing a more general regulatory feature, we have identified other internal sORFs which repress the translation of N-terminally truncated proteoforms. In an in vitro model of myogenic differentiation, the expression of nTRIP6 is transiently upregulated through a mechanistic Target of Rapamycin Complex 1-dependent increase in translation initiation at the internal AUG. Thus, the translation of N-terminally truncated proteoforms can be regulated independently of the canonical ORF.

Keywords:  Myogenesis; Proteoforms; Short ORFs; Translation; Trip6

DOI:  https://doi.org/10.1038/s44319-025-00390-z
Nat Commun. 2025 Feb 18. 16(1): 1706

Non-AUG HIV-1 uORF translation elicits specific T cell immune response and regulates viral transcript expression.

Emmanuel Labaronne, Didier Décimo, Lisa Bertrand, Laura Guiguettaz, Thibault J M Sohier, David Cluet, Valérie Vivet-Boudou, Ana Luiza Chaves Valadão, Clara Dahoui, Pauline François, Isabelle Hatin, Olivier Lambotte, Assia Samri, Brigitte Autran, Lucie Etienne, Caroline Goujon, Jean-Christophe Paillart, Olivier Namy, Bertha Cecilia Ramirez, Théophile Ohlmann, Arnaud Moris, Emiliano P Ricci.

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus that relies on alternative splicing, translational, and post-translational mechanisms to produce over 15 functional proteins from its single ~10 kb transcriptional unit. Using ribosome profiling, nascent protein labeling, RNA sequencing, and whole-proteomics of infected CD4 + T lymphocytes, we characterized the transcriptional, translational, and post-translational landscape during infection. While viral infection exerts a significant impact on host transcript abundance, global translation rates are only modestly affected. Proteomics data reveal extensive transcriptional and post-translational regulation, with many genes showing opposing trends between transcript/ribosome profiling and protein abundance. These findings highlight a complex regulatory network orchestrating gene expression at multiple levels. Viral ribosome profiling further uncovered extensive non-AUG translation of small peptides from upstream open reading frames (uORFs) within the 5' long terminal repeat, which elicit specific T cell responses in people living with HIV. Conservation of uORF translation among retroviruses, along with TAR sequences, shapes DDX3 dependency for efficient translation of the main viral open reading frames.

DOI: https://doi.org/10.1038/s41467-025-56772-3
bioRxiv. 2025 Jan 27. pii: 2025.01.25.634884. [Epub ahead of print]

NCBP2-AS2 is a mitochondrial microprotein, regulates energy metabolism and neurogenesis, and is downregulated in Alzheimer's disease.

Stanislava Popova, Prabesh Bhattarai, Elanur Yilmaz, Daniela Lascu, Juo-Han Kuo, Gizem Erdem, Basak Coban, Jitka Michling, Mehmet Ilyas Cosacak, Huseyin Tayran, Thomas Kurth, Alexandra Schambony, Frank Buchholz, Marc Gentzel, Caghan Kizil.

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimer's disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimer's disease.

DOI: https://doi.org/10.1101/2025.01.25.634884
Cell Death Differ. 2025 Feb 17.

The LINC01315-encoded small protein YAPer-ORF competes with PRP4k to hijack YAP signaling to aberrantly promote cell growth.

Zhu Xie, Chao Li, Rui Huang, Bo Wu, Qian Huang, Zhe Zhang, Tongjin Zhao, Lingqian Wu, Chengtao Li, Jianfeng Shen, Hongyan Wang.

The dysregulation of YAP activity is implicated in abnormal organ size and the pathogenesis of diverse diseases, including cancer. However, the functional regulation of YAP activity by lncRNA-encoded peptides remains elusive. In this study, we report the identification of a small protein (93 aa) encoded by the lncRNA LINC01315. This small protein, termed YAPer-ORF, preferentially interacted with GNAQ/11 mutants to augment YAP activity. Mechanistically, YAPer-ORF was located in the nucleus and competed with YAP to bind the nuclear kinase PRP4K to hinder YAP phosphorylation. This decreased phosphorylation of YAP by YAPer-ORF promoted YAP retention in the nucleus and facilitated the expression of downstream target genes such as CCND1. In both cancerous and noncancerous models, YAPer-ORF prominently drove cell proliferation in a CCND1-dependent manner. Notably, cardiac-specific genetic knock-in of the human YAPer-ORF in mice significantly increased heart size through increased cardiomyocyte proliferation, underscoring the role of YAPer-ORF in cell proliferation. Moreover, treatment with an anti-YAPer-ORF neutralizing antibody effectively suppressed uveal melanoma growth, highlighting the therapeutic potential of targeting YAPer-ORF. These findings collectively establish YAPer-ORF as a critical regulator of YAP activity, further highlighting the disruption of YAPer-ORF activity as a potential therapeutic strategy against YAP-driven human cancers and developmental diseases.

DOI: https://doi.org/10.1038/s41418-025-01449-z