Biology subjects

Hubner, N.

Publications and source records attributed to Hubner, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Loss of cardiomyocyte eukaryotic elongation factor 1 A2 in adult mice triggers cardiomyopathy due to defective proteostasis

Eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to ribosomes but also has additional, non-canonical functions. Mammals express two paralogs: eEF1A1 is ubiquitous, whereas eEF1A2 is confined to adult cardiomyocytes, skeletal myocytes, and neurons. Mutations in EEF1A2 cause cardiomyopathy, but underlying mechanisms remain unclear. Using adult, cardiomyocyte-specific Eef1a2 knock-out (Eef1a2-cKO) and Eef1a1/Eef1a2 double knock-out mice, we show that Eef1a2-cKO animals develop cardiomyopathy with increased mortality, systolic dysfunction, and fibrosis, despite unchanged global protein synthesis, while double knock-out mice die early in a sudden manner. Multi-omics analyses reveal post-transcriptional upregulation of ribosomal proteins and translational regulators in both models. Eef1a2-cKO hearts accumulate autophagosomes and protein aggregates, indicating defective autophagy. Mechanistically, we found that eEF1A2 functions as a chaperone supporting protein folding and proteostasis in cardiomyocytes. Early Rapamycin treatment (mTORC1 inhibition) normalizes systolic heart function and survival in Eef1a2-cKO mice and clears autophagosomes and protein aggregates. Thus, eEF1A2 maintains cardiac proteostasis, and mTORC1 inhibition may represent a therapeutic strategy for patients with EEF1A2 mutations.

physiology↗

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↗

bioRxiv2023

Natural genetic variation quantitatively regulates heart-rate and -dimension

The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In a segregation population of 1192 HO5 x HdrR F2 embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes in loss-of-function models revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart. One-Sentence SummaryKey loci for vertebrate heart function mapped and validated, highlighting diagnostic and potential therapeutic targets for cardiac disorders.

genetics↗

bioRxiv2023

Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

Human ageing is associated with metabolic dysfunction, sarcopenia and frailty that taken together reduce healthspan. For age-associated diseases and lifespan, ERK, AMPK and mTORC1 represent critical pathways, across species1-7. Here we examined the hypothesis that IL11, recently shown to regulate ERK/mTORC1, is an inflammaging factor important for healthspan. As mice age, IL11 is progressively upregulated in liver, skeletal muscle, and fat to stimulate an ERK/AMPK/mTORC1 axis of cellular, tissue- and organismal-level ageing pathologies. In old mice, deletion of Il11 or Il11ra1 protects against metabolic multi-morbidity, sarcopenia, and frailty. Administration of anti-IL11 therapy to elderly mice for six months reactivates an age-repressed program of white fat beiging, reverses metabolic dysfunction, restores muscle function, and reduces frailty. Across studies, inhibition of IL11 lowers epigenetic age, reduces telomere attrition, and preserves mitochondrial function. Towards clinical translation, we generated, humanised, and engineered a neutralising, high-affinity IL11 antibody. These studies identify IL11 as a key inflammaging factor and therapeutic target for mammalian healthspan.

molecular biology↗

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↗

bioRxiv2023

Dominance is common in mammals and is associated with trans-acting gene expression and alternative splicing

BackgroundDominance and other non-additive genetic effects arise from the interaction between alleles, and historically these phenomena played a major role in quantitative genetics. However, today most genome-wide association studies (GWAS) assume alleles act additively. MethodsWe systematically investigated both dominance - here representing any non-additive effect - and additivity across 574 physiological and gene expression traits in three mammalian models: a Pig F2 Intercross, a Rat Heterogeneous Stock and a Mouse Heterogeneous Stock. ResultsIn all species, and across all physiological traits, dominance accounts for about one quarter of the heritable variance. Hematological and immunological traits exhibit the highest dominance variance, possibly reflecting balancing selection in response to pathogens. Although most quantitative trait loci (QTLs) are detectable assuming additivity, we identified 154, 64 and 62 novel dominance QTLs in pigs, rats and mice respectively, that were undetectable as additive QTLs. Similarly, even though most cis-acting eQTLs are additive, we observed a large fraction of dominance variance in gene expression, and trans-acting eQTLs are enriched for dominance. Genes causal for dominance physiological QTLs are less likely to be physically linked to their QTLs but instead act via trans-acting dominance eQTLs. In addition, in HS rat transcriptomes, thousands of eQTLs associate with alternate transcripts and exhibit complex additive and dominant architectures, suggesting a mechanism for dominance. ConclusionsAlthough heritability is predominantly additive, many mammalian genetic effects are dominant and likely arise through distinct mechanisms. It is therefore advantageous to consider both additive and dominance effects in GWAS to improve power and uncover causality.

genetics↗

bioRxiv2023

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators

BackgroundMutation of the PRDM16 gene has been associated with human cardiomyopathy. The PRDM16 protein is a transcriptional regulator affecting cardiac development via Tbx5 and Hand1 regulating myocardial structure. Biallelic Prdm16 inactivation induces severe cardiac dysfunction with postnatal lethality and hypertrophy in mice. Early pathological events upon Prdm16 inactivation have not been explored. MethodsThis study performed in depth pathophysiological and molecular analysis of male and female Prdm16csp1/wt mice carrying systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes with transcriptomics, proteomics, and metabolomics. Kinetic modelling of the cardiac metabolism was undertaken in silico with CARDIOKIN. ResultsPrdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt hearts demonstrate moderate alterations of specific transcripts and protein levels with consistent upregulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre B-cell leukemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional upregulation was detected for Prdm16 itself probably by an autoregulatory mechanism. Prdm16csp1/wt cardiac tissue showed reduction of metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and tricarboxylic acid cycle. Global lipid metabolism was also affected with accumulation of triacylglycerides detected in male Prdm16csp1/wt hearts. In addition, Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. Metabolic modelling in silico suggested lowered fatty acid utilization in male and reduced glucose utilization in female Prdm16csp1/wt cardiac tissue. ConclusionsMonoallelic Prdm16 mutation restricts cardiac performance in Prdm16csp1/wt mice. Metabolic alterations precede transcriptional dysregulation in Prdm16csp1/wt cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype indicating a sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in PRDM16 associated cardiac pathology. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABS- Mutation of the PRDM16 gene has been associated with human cardiomyopathy. - Biallelic inactivation of Prdm16 in mice induces severe cardiac dysfunction with early postnatal lethality. - Prdm16 cooperates with transcription factors such as Tbx5 and Hand1 to activate transcriptional programs that define the development of the compacted myocardium. What New Information Does This Article Contribute?- Systemic, monoallelic inactivation in Prdm16csp1/wt mice induces cardiac dysfunction with normal survival. - Metabolic alterations are the leading pathophysiological consequences and induce cardiac hypoplasia. On the molecular level this is associated with upregulation of metabolic regulators Pyroxd2 and Pbxip1. - Metabolic response after Prdm16 inactivation occurs in a sex specific manner.

pathology↗

bioRxiv2022

Coronary vessel assembly involves patterned endocardial sprouting and tip-cell-to artery specification

The coronary vasculature comprises superficial coronary veins and deeper coronary arteries and capillaries that critically support the high metabolic activity of the beating heart. Understanding how different endothelial progenitor sources jointly shape and remodel the coronary vasculature into its adult pattern has recently attracted great research interest, and spurred much debate. Here, using lineage tracing tools in combination with three-dimensional imaging, live-imaging in explants and single-cell transcriptional profiling, we demonstrate that sprouting angiogenesis drives both sinus venosus and endocardial contribution to coronary plexus. Whereas previous studies limit endocardial contribution to coronary vessels to the interventricular septum and ventral aspect of the heart, our study demonstrates extensive endocardial sprouting angiogenesis in the free ventricular walls. In particular, we identify a distinct subpopulation of endocardial cells that express future coronary markers and reside in both the embryonic and adult ventricular wall endocardium. Most importantly, we provide evidence for sprouting angiogenesis from both endocardium and subepicardial plexus towards the inner myocardial wall to determine pre-arterial specification. Additionally, sprouting from the endocardium leads to the establishment of perfused connections to the advancing coronary plexus, also followed by transitioning to the pre-arterial cell state. Distinct molecular profiles characterize sprouting populations in the intramyocardial and subepicardial layers that shape the prospective coronary arteries and veins, respectively. Harnessing the endocardial progenitors and targeting the distinct sprouting populations may in the future serve to tailor cardiac vascular adaptations for therapeutic purposes.

developmental biology↗

bioRxiv2022

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutieres syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here we show that SAM domain and HD domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

immunology↗

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

Identification of novel disease relevant genetic modifiers affecting the SHH pathway in the developing brain

Pathogenic gene variants in humans affecting the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown genetic modifiers. To identify such modifiers, we established novel congenic mouse models. LRP2 deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on FVB/N background indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are new positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized PTTG1 as a novel primary cilia component in the neuroepithelium. The identification of genes, that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart, is likely relevant to understand variability in human congenital disorders.

developmental 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↗