Biology subjects

Martinez, T. F.

Publications and source records attributed to Martinez, T. F..

8 recordsLinked to original sources

bioRxiv2026

HDAC5-encoded Microprotein NISM Mediates Nucleolar Formation and Ribosomal RNA Synthesis

Ribosome biogenesis is the process by which ribosomal RNA (rRNA) and ribosomal proteins are synthesized, processed, and assembled into functional ribosomes. This process begins in the nucleolus, a multiphase liquid condensate. Here, we discover an arginine-rich disordered nucleolar microprotein encoded within the HDAC5 5'-UTR that we termed Nucleolar Integrity and Stress Microprotein (NISM). NISM overexpression leads to impaired rDNA transcription, triggering nucleolar stress, p53 activation, and suppressed proliferation. NISM knockout causes disruption of nucleolar structure and also induces p53 activation. Mechanistically, NISM interacts with the DExH-box RNA helicase DHX9 and regulates its activities related to pre-rRNA synthesis. Computational analyses and polymer physics-based mathematical modeling revealed that NISM coordinates nucleolar formation and pre-rRNA synthesis by enhancing the liquid-liquid phase separation of DHX9. This study establishes NISM as a regulator of nucleolar biology and deepens our understanding of how disordered microproteins can facilitate the formation of membraneless organelles.

molecular biology↗

bioRxiv2025

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

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.

genomics↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2024

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

molecular biology↗

bioRxiv2024

Sex-dependent metabolic remodeling of kidneys revealed by arteriovenous metabolomics

Sex is a fundamental biological variable important in biomedical research, drug development, clinical trials, and prevention approaches. Among many organs, kidneys are known to exhibit remarkable structural, histological, and pathological differences between sexes. However, whether and how kidneys display distinct metabolic activities between sexes is poorly understood. By developing kidney-specific arteriovenous (AV) metabolomics combined with transcriptomics, we report striking sex differences in both basal metabolic activities and adaptive metabolic remodeling of kidneys after a fat-enriched ketogenic diet (KD), a regimen known to mitigate kidney diseases and improve immunotherapy for renal cancer. At the basal state, female kidneys show highly accumulated aldosterone and various acylcarnitines. In response to the KD, aldosterone levels remain high selectively in females but the sex difference in acylcarnitines disappears. AV data revealed that, under KD, female kidneys avidly take up circulating fatty acids and release 3-hydroxybutyrate (3-HB) whereas male kidneys barely absorb fatty acids but consistently take up 3-HB. Although both male and female kidneys take up gluconeogenic substrates such as glycerol, glutamine and lactate, only female kidneys exhibit net glucose release. Kidney transcriptomics data incompletely predict these sex differences, suggesting post-transcriptional/translational regulation mechanisms. This study provides foundational insights into the sex-dependent and diet-elicited metabolic flexibility of the kidneys in vivo, serving as a unique resource for understanding variable disease prevalence and drug responses between male and female kidneys.

biochemistry↗

bioRxiv2023

Comparison of software packages for detecting unannotated translated small open reading frames by Ribo-seq

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess ORF translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH), and found surprisingly low agreement across all tools. Only [~]2% of smORFs were called translated by all five tools and [~]15% by three or more tools when assessing the same high- resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed [~]72% agreement across all five tools. We also found that some tools are strongly biased against low- resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage as a proxy for translation levels revealed that highly translated smORFs are more likely to be detected by more than one tool. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs, and choosing the tools based on the quality of the dataset and planned downstream characterization experiments of predicted smORFs.

genomics↗

bioRxiv2022

Profiling Mouse Brown and White Adipocytes to Identify Metabolically Relevant Small ORFs and Functional Microproteins

The absence of thousands of recently annotated small open reading frame (smORF)-encoded peptides and small proteins (microproteins) from databases has precluded their analysis in metabolism and metabolic disease. Given the outsized importance of small proteins and peptides such as insulin, leptin, amylin, glucagon, and glucagon-like peptide-1 (GLP-1) in metabolism, microproteins are a potentially rich source of uncharacterized metabolic regulators. Here, we annotate smORFs in primary differentiated brown, white, and beige mouse adipose cells. Ribosome profiling (Ribo-Seq) detected a total of 3,877 unannotated smORFs. Analysis of RNA-Seq datasets revealed diet-regulated smORF expression in adipose tissues, and validated the adipose translation of the feeding-neuron marker gene Gm8773. Gm8773 encodes the mouse homolog of FAM237B, a neurosecretory protein that stimulates food intake and promotes weight gain in chickens. Testing of recombinant mFAM237B produced similar orexigenic activity in mice further supporting a role for FAM237B as a metabolic regulator and potentially part of the brain-adipose axis. Furthermore, we demonstrated that data independent acquisition mass spectrometry (DIA-MS) proteomics can provide a sensitive, flexible, and quantitative platform for identifying microproteins by mass spectrometry. Using this system led to the detection of 58 microproteins from cell culture and an additional 33 from mouse plasma. The proteomics data established the anti-inflammatory microprotein AW112010 as a circulating factor, and found that plasma levels of a microprotein translated from a FRS2 uORF is elevated in older obese mice. Together, the data highlight the value of this database in examining understudied smORFs and microproteins in metabolic research and identifying additional regulators of metabolism.

systems biology↗

bioRxiv2019

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

genomics↗