Biology subjects

van Heesch, S.

Publications and source records attributed to van Heesch, S..

13 recordsLinked to original sources

bioRxiv2025

LINC01503-MP is a Mitochondrial Microprotein That Promotes Cell Proliferation and Oxidative Metabolism.

Long non-coding RNAs (lncRNAs) are well-established as key regulators of gene expression. However, emerging evidence reveals that some lncRNAs can also encode functional microproteins. In this study, we report the identification of an evolutionarily young microprotein encoded by LINC01503, expressed across several human tissues. This microprotein, designated as LINC01503-MP, localises to the mitochondria and exerts a proliferative effect on HCT116 colorectal cancer (CRC) cells. Functional studies reveal that LINC01503-MP regulates mitochondrial oxygen consumption rate, linking its activity to enhanced metabolic functions and cell proliferation. Interactome analyses identified multiple mitochondrial metabolism-related proteins as potential interaction partners. Our findings show that LINC01503-MP plays a role in the proliferative phenotype associated with LINC01503 upregulation in CRC, suggesting the functional significance of evolutionarily young, lncRNA-derived microproteins in cancer progression.

molecular biology↗

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↗

bioRxiv2023

What can Ribo-seq and proteomics tell us about the non-canonical proteome?

Ribosome profiling (Ribo-seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of non-canonical sites of ribosome translation outside of the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7,000 non-canonical open reading frames (ORFs) are translated, which, at first glance, has the potential to expand the number of human protein-coding sequences by 30%, from [~]19,500 annotated CDSs to over 26,000. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of non-canonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome, but searching for guidance on how to proceed. Here, we discuss the current state of non-canonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein-coding". In briefThe human genome encodes thousands of non-canonical open reading frames (ORFs) in addition to protein-coding genes. As a nascent field, many questions remain regarding non-canonical ORFs. How many exist? Do they encode proteins? What level of evidence is needed for their verification? Central to these debates has been the advent of ribosome profiling (Ribo-seq) as a method to discern genome-wide ribosome occupancy, and immunopeptidomics as a method to detect peptides that are processed and presented by MHC molecules and not observed in traditional proteomics experiments. This article provides a synthesis of the current state of non-canonical ORF research and proposes standards for their future investigation and reporting. HighlightsO_LICombined use of Ribo-seq and proteomics-based methods enables optimal confidence in detecting non-canonical ORFs and their protein products. C_LIO_LIRibo-seq can provide more sensitive detection of non-canonical ORFs, but data quality and analytical pipelines will impact results. C_LIO_LINon-canonical ORF catalogs are diverse and span both high-stringency and low-stringency ORF nominations. C_LIO_LIA framework for standardized non-canonical ORF evidence will advance the research field. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/541049v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@4878d0org.highwire.dtl.DTLVardef@1f3c9e4org.highwire.dtl.DTLVardef@4ba1f7org.highwire.dtl.DTLVardef@17695a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets. HighlightsO_LIRibo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma C_LIO_LIHigh-resolution CRISPR tiling reveals uORF functions in medulloblastoma C_LIO_LIASNSD1-uORF controls downstream pathways with the prefoldin-like complex C_LIO_LIASNSD1-uORF is necessary for medulloblastoma cell survival C_LI

cancer biology↗

bioRxiv2022

Aging-regulated TUG1 is dispensable for endothelial cell function

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

cell biology↗

bioRxiv2021

RPL3L-containing ribosomes modulate mitochondrial activity in the mammalian heart

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse specialized ribosomes is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralog of RPL3 (uL3) that is exclusively expressed in muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes upregulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-Seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L neither modulates translational efficiency nor ribosome affinity towards a specific subset of transcripts. By contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of mitochondrial activity fine-tuning. Our results demonstrate that the existence of tissue-specific RP paralogs does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.

molecular biology↗

bioRxiv2021

Cap analysis of gene expression (CAGE) sequencing reveals alternative promoter usage in complex disease

The role of alternative promoter usage in tissue specific gene expression has been well established, however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) tag sequencing from the left ventricle (LV) of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, the Brown Norway (BN) to understand role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites (TSS) in the rat LV, including 1,970 novel cardiac TSS resulting in new transcripts. We identified 27 genes with alternative promoter usage between SHR and BN which could lead to protein isoforms differing at the amino terminus between two strains. Additionally, we identified 475 promoter switching events where a shift in TSS usage was within 100bp between SHR and BN, altering length of the 5 UTR. Genomic variants located in the shifting promoter regions showed significant allelic imbalance in F1 crosses, confirming promoter shift. We found that the insulin receptor gene (Insr) showed a switch in promoter usage between SHR and BN in heart and liver. The Insr promoter shift was significantly associated with insulin levels and blood pressure within a panel of BXH/HXB recombinant inbred (RI) rat strains. This suggests that the hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

bioinformatics↗

bioRxiv2021

A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational vocabulary of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

genomics↗

bioRxiv2021

Dual-function RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes.

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Similar to the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8 - all identified as multifunctional RBPs by our analysis - selectively influence target translation rates depending on 5 UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.

systems biology↗

bioRxiv2020

Trans control of cardiac mRNA translation in a protein length-dependent fashion

Little is known about the impact of naturally occurring genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate how genetic influences on mRNA translational efficiency are associated with complex disease phenotypes using a panel of rat recombinant inbred lines. We identify a locus for cardiac hypertrophy that is associated with a translatome-wide and protein length-dependent shift in translational efficiency. This master regulator primarily affects the translation of very short and very long protein-coding sequences, altering the physiological stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus points to altered ribosome assembly, characterized by accumulation of polysome half-mers, changed ribosomal configurations and misregulation of the small nucleolar RNA SNORA48. We postulate that this locus enhances a pre-existing negative correlation between protein length and translation initiation in diseased hearts. Our work shows that a single genomic locus can trigger a complex, translation-driven molecular mechanism that contributes to phenotypic variability between individuals. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/133298v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@e46986org.highwire.dtl.DTLVardef@1cc0b08org.highwire.dtl.DTLVardef@e86011org.highwire.dtl.DTLVardef@1fb3706_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGenetic variability impacts protein synthesis rates in a rat model for cardiac hypertrophy C_LIO_LIA trans locus affects stoichiometric translation rates of cardiac sarcomeric proteins C_LIO_LIThis master regulator locus induces a global, protein length-dependent shift in translation C_LIO_LIDysregulated ribosome assembly induces half-mer formation and affects translation initiation rate C_LI

genetics↗

bioRxiv2020

A functional screen of translated pancreatic lncRNAs identifies a microprotein-independent role for LINC00261 in endocrine cell differentiation

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Here, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated during pancreas development. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for endocrine cell differentiation. However, deletion of LINC00261 diminishes generation of insulin+ endocrine cells, in a manner independent of the nearby TF FOXA2. Systematic deletion of each of LINC00261s seven poorly conserved microproteins shows that the RNA, rather than the microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs into recently evolved microproteins during human pancreas development and provides a blueprint for dissection of their coding and noncoding roles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/062679v1_ufig1.gif" ALT="Figure 1"> View larger version (96K): org.highwire.dtl.DTLVardef@10715caorg.highwire.dtl.DTLVardef@27af76org.highwire.dtl.DTLVardef@17083a6org.highwire.dtl.DTLVardef@1d31f9f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIExtensive lncRNA translation and microprotein production during human pancreas development C_LIO_LIA small-scale loss-of-function screen shows most translated lncRNAs are dispensable C_LIO_LILINC00261 is highly translated and regulates endocrine cell differentiation C_LIO_LIDeleting LINC00261s evolutionary young microproteins reveals no essential roles C_LI

developmental biology↗

bioRxiv2019

The Tug1 Locus is Essential for Male Fertility

BackgroundSeveral long noncoding RNAs (lncRNAs) have been shown to function as central components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, their functions remain largely uncharacterized. This is compounded by the fact that identifying lncRNA loci that have robust and reproducible phenotypes when mutated has been a challenge. ResultsWe previously generated a cohort of 20 lncRNA loci knockout mice. Here, we extend our initial study and provide a more detailed analysis of the highly conserved lncRNA locus, Taurine Upregulated Gene 1 (Tug1). We report that Tug1 knockout male mice are sterile with complete penetrance due to a low sperm count and abnormal sperm morphology. Having identified a lncRNA loci with a robust phenotype, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region (DNA, RNA, protein, or the act of transcription) have activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors three distinct regulatory activities - two noncoding and one coding: (i) a cis DNA repressor that regulates many neighboring genes, (ii) a lncRNA that can regulate genes by a trans-based function, and finally (iii) Tug1 encodes an evolutionary conserved peptide that when overexpressed impacts mitochondrial membrane potential. ConclusionsOur results reveal an essential role for the Tug1 locus in male fertility and uncover three distinct regulatory activities in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

genomics↗

bioRxiv2018

Translational control of cardiac fibrosis

Abstract BackgroundFibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored.\n\nMethodsGenome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used miRNA-and RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. To reveal post-transcriptional mechanisms in the human fibrotic heart, we then integrated our findings with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients.\n\nResultsWe generated nucleotide-resolution translatome data during the TGF{beta}1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested an extensive post-transcriptional regulatory network underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts.\n\nConclusionsWe reveal widespread translational effects of TGF{beta}1 and define novel post-transcriptional events that control the fibroblast-to-myofibroblast transition. Regulatory networks that affect ribosome occupancy in fibroblasts are paralleled in human heart disease. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.

genomics↗