Biology subjects

Valen, E.

Publications and source records attributed to Valen, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Oncogenic fusions induce an extensive cancer-restricted cryptic proteome in Ewing sarcoma

How tumors generate a cryptic "dark" proteome absent from healthy tissues, and whether it can be reversibly activated, remains unclear. In Ewing sarcoma, all tumors are driven by EWSR1::ETS fusions, making it a tractable model to study dark proteome activation. Integrating matched transcriptomes, translatomes and proteomes from 48 patient tumors with long-read RNA sequencing, single-cell Ribo-seq, proteomics and immunopeptidomics in cell line models, we show that EWSR1::FLI1 acts as a reversible switch recurrently inducing hundreds of cancer-specific neoproteins. Many arise from canonical coding regions via intragenic transcription start sites, generating truncated or out-of-frame neoproteins. We uncover a fusion-dependent increase in ribosomal readthrough into poly(A) tails, generating a stable 80-amino-acid TRPM4 neoprotein that, despite lacking a stop codon, is the most abundant tumor-specific microprotein across patients. Proteomic, immunopeptidomic, and immunofluorescence analyses validate neoproteins as tumor-restricted antigens, revealing a therapeutically actionable cancer dark proteome controlled by one oncogenic fusion.

cancer biology↗

bioRxiv2026

Ribosome profiling to study translation in cortical synaptoneurosomes on group 1 mGluR activation

Local protein synthesis in neurons occurs in both axons and dendrites and plays a central role in synaptic function. High-throughput-based sequencing and imaging studies have demonstrated the presence and translation of synaptically localised mRNAs. However, quantification of activity-dependent translation dynamics at synapses at the transcriptome-wide scale remains limited. Here, we apply ribosome profiling to synapse-enriched fractions (synaptoneurosomes) derived from rat cortical tissue following stimulation with the group 1 mGluR agonist DHPG. DHPG stimulation induced translation of mRNAs involved in synaptic processes, including synaptic vesicle exocytosis and axo-dendritic transport. Notably, translation of ribosomal protein mRNAs was upregulated upon mGluR activation, consistent with the expected increase in de novo protein synthesis. Together, these results demonstrate the use of ribosome profiling to capture changes in local mRNA translation from isolated preparations.

neuroscience↗

bioRxiv2026

Decoding the Sequence Requirements for Translation Initiation

Accurate selection of start codons by ribosomes is a fundamental determinant of proteome composition. Although the Kozak sequence--an 8-nucleotide sequence flanking the start codon--has long been viewed as the primary determinant of initiation in eukaryotes, it fails to explain the large diversity of start codon usage across transcripts. Here we combine massively parallel reporter assays, bioinformatics, machine learning, single-molecule imaging and cryo-electron microscopy to define the extended translation initiation sequence (eTIS), an [~]80-nucleotide sequence surrounding the start codon that governs initiation efficiency. A deep-learning model trained on eTIS features accurately predicts translation initiation across transcripts. Unexpectedly, we find that the Kozak sequence is not optimal for initiation as is widely presumed, and we identify the origin of this discrepancy. eTIS nucleotides that promote efficient initiation are enriched in the human transcriptome and are evolutionarily conserved, underscoring their functional importance. Biophysical and structural analyses reveal that specific eTIS residues--including the key +6 position and residues in the mRNA entry and exit channel--engage ribosomal proteins, rRNA and initiation factors to promote start codon recognition by stabilizing the ribosome at the start codon and facilitating the structural transitions required for initiation. Finally, optimization of the eTIS markedly enhances translational fidelity and protein output from therapeutic mRNAs, highlighting its practical utility. Together, these findings redefine the sequence logic of translation initiation and establish a framework for precise control of protein expression.

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

CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detection

CRISPR/Cas systems offer powerful tools for genome editing, but their therapeutic application is hampered by the risk of unintended off-target mutations. Many molecular methods have been established to detect off-target editing, however, their sensitivity depends on first identifying potential sites using in silico methods. However, these in silico prediction methods are challenged by a trade-off between speed and sensitivity, and can fail to comprehensively detect all edited off-target sites. Here, we demonstrate that ignoring bulges has led to missing editing at off-target sites in previous studies and that continuing this practice can lead to inflated claims of fidelity. As a solution, we introduce the concept of symbolic alignments to efficiently identify off-targets without sacrificing sensitivity. We further present specialized data structures that enable rapid, alignment-free probabilistic ranking of guide RNAs based on their predicted off-target burden. Implemented in the tool CHOPOFF, these innovations accommodate mismatches, bulges (insertions/deletions), and incorporate genomic sequence variants for personalized off-target assessment. Benchmarking demonstrates that CHOPOFF significantly outperforms state-of-the-art tools in both prediction accuracy and computational speed. AvailabilityCHOPOFF command line available at https://github.com/JokingHero/CHOPOFF.jl CHOPOFF web server available at https://crisprtools.org/chopoff

bioinformatics↗

bioRxiv2024

Nano3P-seq: charting the coding and non-coding transcriptome at single molecule resolution

RNA polyadenylation is crucial for RNA maturation, stability and function, with polyA tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3 end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances, tail composition and tail length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA molecule from its 3 end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flowcells, as well as compatible software for polyA tail length and content prediction, which we term PolyTailor. We demonstrate that PolyTailor provides accurate estimates of transcript abundances, tail lengths and content information, while capturing both coding and non-coding RNA biotypes, including mRNAs, snRNAs, and rRNAs. This method can be applied to any RNA sample of interest (e.g. poly(A)-selected, ribodepleted, total RNA), and can be completed in one day. The Nano3P-seq protocol can be performed by researchers with moderate experience in molecular biology techniques and nanopore sequencing library preparation, and basic knowledge of linux bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and non-coding transcriptome in a comprehensive and reproducible manner. Key PapersBe[g]ik O, Diensthuber G, Liu H, Delgado-Tejedor A, Kontur C, Niazi AM, Valen E, Giraldez AJ, Beaudoin JD, Mattick JS, Novoa EM. Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods 20, 75-85 (2023). https://doi.org/10.1038/s41592-022-01714-w Delgado-Tejedor A, Medina M, Begik O, Cozzuto L, Lopez J, Blanco B, Ponomarenko J, Novoa EM. Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites. NatComm 15, 10054 (2024). https://doi.org/10.1038/s41467-024-54368-x

molecular biology↗

bioRxiv2024

Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain

Protein synthesis in neurons is highly compartmentalised and regulated, with key roles for translation initiation and elongation factors. The most widely used transcriptome-wide method for measuring translation, ribosome profiling, characterises the elongation phase of translation but does not provide insight into the initiation phase with scanning of the small ribosomal subunit (SSU). Here, we adapted and optimised ribosome complex profiling (RCP-seq) for brain tissue, capturing SSUs and analysis of translation initiation dynamics in mouse dentate gyrus and cerebral cortex. In both tissues, SSUs accumulate upstream of the start codon on synaptically localised RNAs and this poised SSU configuration is associated with enhanced translational efficiency. Upstream open reading frames (uORFs) are extensively translated and associated with less SSU poising downstream, suggesting that uORFs may have a buffering effect on poised SSUs. Ribosome occupancy analysis suggests that neuron-specific transcripts recruit more ribosomes and are more translated than glia-specific transcripts. Furthermore, monosome-preferring neuronal mRNAs exhibit reduced scanning and elongation relative to polysome-preferring transcripts implying reduced recruitment of ribosomes. In sum, RCP-seq elucidates translation initiation dynamics in the mammalian brain and uncovers cell-type- and transcript-specific regulation.

neuroscience↗

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

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗

bioRxiv2024

Reprograming gene expression in hibernating C. elegans involves the IRE-1/XBP-1 pathway

In nature, many animals respond to cold by entering hibernation, while in clinical settings, controlled cooling is used in transplantation and emergency medicine. However, the molecular mechanisms that enable cells to survive severe cold are still not fully understood. One key aspect of cold adaptation is the global downregulation of protein synthesis. Studying it in the nematode Caenorhabditis elegans, we find that the translation of most mRNAs continues in the cold, albeit at a slower rate, and propose that cold-specific gene expression is regulated primarily at the transcription level. Supporting this idea, we found that the transcription of certain cold-induced genes is linked to the activation of unfolded protein response (UPR) through the conserved IRE-1/XBP-1 signaling pathway. Our findings suggest that this pathway is triggered by cold-induced perturbations in proteins and lipids within the endoplasmic reticulum, and that its activation is beneficial for cold survival.

cell biology↗

bioRxiv2023

Comprehensive contact tracing during an outbreak of alpha-variant SARS-CoV-2 in a rural community reveals less viral genomic diversity and higher household secondary attack rates than expected

Sequencing of SARS-CoV-2 genomes throughout the COVID-19 pandemic has generated a wealth of data on viral evolution across populations, but only a few studies have so far explored SARS-CoV-2 evolution across transmission networks of tens to hundreds of persons. Here, we couple data from SARS-CoV-2 sequencing with contact tracing data from an outbreak with a single origin in a rural Norwegian community where samples from all exposed persons were collected prospectively. A total of 134 nasopharyngeal samples were positive by PCR. Among the 121 retrievable genomes, 81 were identical to the genome of the introductor, thus demonstrating that genomics offers limited additional value to manual contact-tracing. In the cases where mutations were discovered, five small genetic clusters were identified. We observed a household secondary attack rate of 67%, with 92% of household members infected among households with secondary transmission, suggesting that SARS-CoV-2 introduction into large families are likely to affect all household members. ImportanceIn outbreak investigations, obtaining a full overview of infected individuals within a population is seldom acheived. We here present an example of just that, where a single introduction of B1.1.7 SARS-CoV-2 within a rural community allowed for tracing of the virus, from an introductor via dissemination through larger gatherings, into households. The outbreak occurred before widespread vaccination, allowing for a "natural" outbreak development with community lock-down. We show through sequencing that the virus can infect up to five consecutive persons without gaining mutations, thereby showing that contact tracing seems more important than sequencing for local outbreak investigations. We also show how families with small children are less likely to contain spread to all family members if SARS-CoV-2 enters the household either by a child or a caregiver, as isolation of the primary infected is difficult in such scenarios.

microbiology↗

bioRxiv2023

Ribosome Decision Graphs for the Representation of Eukaryotic RNA Translation Complexity

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later translons. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.

genomics↗

bioRxiv2021

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

molecular biology↗

bioRxiv2021

ORFik: a comprehensive R toolkit for the analysis of translation

* BackgroundWith the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. * ResultsHere, we introduce ORFik, a user-friendly R/Bioconductor toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames. As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5 UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. * Availabilityhttp://bioconductor.org/packages/ORFik

bioinformatics↗

bioRxiv2020

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary considerably depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the required time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pause and stall sites, but due to library-specific biases, these predictions are often unreliable. Here, we address this by taking advantage of the deep conservation of the protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important positions of ribosome slowdown - here collectively called stall sites. We analyze multiple ribosome profiling datasets from a phylogenetically diverse group of eukaryotes: yeast, fruit fly, zebrafish, mouse, and human and identify conserved stall sites. We find thousands of stall sites across multiple species, with proline, glycine, and negatively charged amino acids being the main facilitators of stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved regulatory effect on RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

genomics↗

bioRxiv2020

Single molecule structure sequencing reveals RNA structural dependencies, breathing and ensembles

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

molecular biology↗

bioRxiv2019

Deconstructing the individual steps of vertebrate translation initiation

Translation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control 1. Despite this centrality the series of steps involved in this process are poorly understood 2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5 UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.

molecular biology↗

bioRxiv2019

Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation

In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remains poorly understood. Here, we generated chromatin accessibility maps from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hyper-transcribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

developmental biology↗