Biology subjects

Cenik, C.

Publications and source records attributed to Cenik, C..

At least 19 recordsLinked to original sources

bioRxiv2026

RiboFlow v2: a configurable ribosome profiling pipeline reveals measurements sensitive to read-mapping methodology

Ribosome profiling experiments capture a snapshot of translation by sequencing ribosome-protected fragments (RPFs). Alignment to a well-curated transcriptome captures most RPF signal, but can miss ribosome activity when expressed exons are absent from the reference. Genome alignment of RPFs expands the detectable sequence space, but can expose ambiguity between homologous loci. Existing workflows often couple reference choice with the handling of ambiguously mapped reads, making it difficult to isolate the effects of either decision on downstream analyses. Here, we developed RiboFlow v2 by adding genome alignment and configurable handling of multimapping reads to the existing transcriptome workflow. Using 24 human ribosome profiling libraries and their matched RNA-seq data, we found that nucleotide-level coverage at matched coding-sequence positions and translation efficiency (TE) were broadly concordant between alignment strategies under stringent alignment filters. Even under these conditions, individual genes showed substantial differences in coverage and TE estimates. These differences included coverage in alternative exons absent from selected transcripts and ambiguous alignments between protein-coding genes and processed pseudogenes. Clustering genes by read-assignment patterns identified groups associated with pseudogene prevalence and highlighted shared exonic sequence among reference transcripts as a source of alignment discrepancies. RiboFlow v2 extends the analysis beyond selected reference transcripts and enables identification of genes whose ribosome coverage and abundance estimates depend on reference composition and how ambiguously mapped reads are handled.

bioinformatics↗

bioRxiv2026

Allele-specific expression is associated with lower translational efficiency in mouse embryos and stem cells

An mRNA's cytoplasmic fate can be shaped by its transcriptional history, its modifications and associated protein complexes. Allele-specific expression (ASE), in which a gene's two alleles are transcribed unequally, provides a natural experiment given that the alleles can differ in sequence or chromatin state, yet their transcripts share the same cytoplasm. We previously found lower translational efficiency (TE) among genes with ASE in early mouse embryos. Because that observation came from one cross direction and from preimplantation stages only, sequence-dependent and parent-of-origin-dependent ASE could not be separated, and whether the association held in other cell types remained unknown. Here we addressed both by generating RNA sequencing and ribosome profiling data from reciprocal-cross mouse 8-cell embryos, individual embryonic stem cell clones, neural progenitor cells, and adult tissues. In 8-cell embryos, both sequence-dependent and parent-of-origin-dependent ASE were associated with lower TE. Genes with ASE at the 8-cell stage also had lower protein abundance at the subsequent morula stage. Ribo-ITP, which enables ribosome profiling from single clones, allowed assessing the relationship between ASE and TE in individual embryonic stem cell clones. We found that TE was lower wherever a gene was allelically imbalanced, and the reduction was larger where that imbalance was consistent across clones. The same association held in neural progenitors, and when ASE was defined from independent published datasets, but was not detected in adult tissues. Allelic imbalance in transcription is therefore accompanied by reduced translation of the resulting mRNAs, though the intermediate carrying allelic information to the translation machinery remains unidentified.

molecular biology↗

bioRxiv2026

Deep mutational scan of the pore of the cold-sensing TRPM8 channel

Members of the Transient Receptor Potential (TRP) family of ion channels have a nearly ubiquitous role in human physiology, tuning cell signaling to remarkably diverse physical and chemical stimuli. Although there is extensive structural data on TRP channels, a systematic and unbiased interrogation of structure-function relations in these proteins is required to fully elucidate their mechanisms of function. By focusing on a critical pore region of the TRPM8 channel, which is the main detector of cold and cooling agents in sensory neurons, we show how deep mutational scanning can be used in combination with the available structural data to understand how TRP channels respond to stimuli. We define a novel mechanism whereby the extracellular pore loop, which has only been resolved in structures representing desensitized states of the channel, plays an essential role in the response of TRPM8 to menthol or cold by coordinating the movement of the S6 helices that line and gate the pore, and the ion-selectivity filter that binds permeant cations. Moreover, our screen reveals sequence determinants along the S6 helices that explain how their architecture sustains gating and, together, provide strong support for a structural mechanism of TRPM8 pore opening in response to menthol and cold.

biophysics↗

bioRxiv2025

Developmental and transcriptional programs that define the initiation of the forelimb

The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq, and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic LPM and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5 binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.

developmental biology↗

bioRxiv2025

A late cytoplasmic surveillance pathway ensures ribosome integrity

Errors in ribosome assembly can produce defective subunits that can lead to aberrant translation events. How faulty ribosomes are recognized and whether they are recognized during assembly or translation remains poorly understood. We utilized a mutation in the ribosomal protein uL16 to track defective 60S subunits through the biogenesis and translation pathways. This mutation deletes a critical loop in the P site and arrests pre- 60S particles during late cytoplasmic maturation. However, simultaneous mutations in the late biogenesis factors Nmd3 and Tif6 bypass this block, releasing defective ribosomes into the translational pool. Cryo-EM and selective ribosome profiling reveal that these ribosomes can form peptide bonds, but stall predominantly at the first few codons. We show that the uL16 mutant ribosomes are detected and targeted for degradation during biogenesis and that they escape degradation if they enter translation. We identify Reh1 as a non-canonical ribosome assembly factor that is required for this surveillance pathway.

molecular biology↗

bioRxiv2025

Ribo-ITP expands the translatome of limited input samples

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can act as regulatory elements or encode functional micropeptides. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this input limitation using Ribo-ITP on difficult-to-collect samples such as microdissected hippocampal tissues and single preimplantation embryos to identify thousands of translons. To test the translational capacity of the identified translons, we engineered a translon-dependent GFP reporter system and detected expression of translons initiating at ATG and near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that may negatively impact growth. We identified distinct expression patterns of translons using a comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types. Further, using a machine learning model we predict that specific upstream translons in synaptically enriched mRNAs regulate translation efficiency of the annotated coding region. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.

genomics↗

bioRxiv2025

Translational buffering tunes gene expression in mouse and human

BackgroundTranslational buffering refers to the regulation of ribosome occupancy to offset the effects of transcriptional variation. While previous studies have primarily investigated translational buffering in yeast under genetic variation or environmental stress, it remains unclear how widespread this is across mammalian genes in various cellular contexts. ResultsWe performed a uniform analysis of 1,515 matched ribosome profiling and RNA-seq datasets from humans and mice. This resource enabled us to assess translational buffering through comparative analysis of variation in ribosome occupancy and RNA expression, and by examining the relationship between mRNA abundance and translation efficiency. We found that translational buffering is partly conserved between humans and mice; homologous genes showed moderate cross-species correlation in mRNA-translation efficiency relationships and strong enrichment of shared buffered genes, particularly those encoding ribosomal, RNA-binding, and proteasomal proteins. Although identified buffered genes associate with specific sequence features, these alone are insufficient to predict translational buffering, highlighting the importance of cellular context. Genes exhibiting translational buffering show lower variation in protein abundance in cancer cell lines and tissues. We also observed that translationally buffered genes are more likely to be haploinsufficient and triplosensitive, suggesting a demand for stringent dosage limits. ConclusionsWe hypothesize two models of translational buffering, namely the "differential accessibility model" and the "translation initiation rate model", suggesting that different transcripts align with one or the other. Our study explores the translational buffering potential of genes across diverse conditions, elucidates their distinctive features, and provides insights into the mechanisms driving this effect.

bioinformatics↗

bioRxiv2025

Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans

Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways-including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)-as indicated by absence of changes in RPS-10 ubiquitination, eIF2 phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.

genetics↗

bioRxiv2024

Landscape and regulation of new protein translation in the early C. elegans embryo

Animal embryos rely on regulated translation of maternally deposited mRNAs to drive early development. Using low-input ribosome profiling combined with RNA sequencing on precisely staged embryos, we measured mRNA translation during the first four cell cycles of C. elegans development. We uncovered stage-specific patterns of developmentally coordinated translational regulation. We confirmed that mRNA localization correlates with translational efficiency, though initial translational repression in germline precursors occurs before P-granule association. Our analysis suggests that the RNA-binding protein OMA-1 represses the translation of its target mRNAs in a stage-specific manner, while indirectly promoting the translational efficiency of other transcripts. These findings illuminate how post-transcriptional mechanisms shape the embryonic proteome to direct cell differentiation, with implications for understanding similar regulation across species where maternal factors guide early development.

developmental biology↗

bioRxiv2024

Translation efficiency covariation across cell types is a conserved organizing principle of mammalian transcriptomes

Characterizing shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and novel biological functions. However, it is unclear if such coordination extends to translation. Here, we uniformly analyzed 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of translation efficiency covariation (TEC), identifying coordinated translation patterns across cell types. We nominate candidate mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and uncovers gene functions that are not evident from RNA or protein co-expression. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish TEC as a conserved organizing principle of mammalian transcriptomes.

bioinformatics↗

bioRxiv2024

Predicting the translation efficiency of messenger RNA in mammalian cells

The degree to which translational control is specified by mRNA sequence is poorly understood in mammalian cells. Here, we constructed and leveraged a compendium of 3,819 ribosomal profiling datasets, distilling them into a transcriptome-wide atlas of translation efficiency (TE) measurements encompassing >140 human and mouse cell types. We subsequently developed RiboNN, a multitask deep convolutional neural network, and classic machine learning models to predict TEs in hundreds of cell types from sequence-encoded mRNA features, achieving state-of-the-art performance (r=0.79 in human and r=0.78 in mouse for mean TE across cell types). While the majority of earlier models solely considered 5' UTR sequence1, RiboNN integrates contributions from the full-length mRNA sequence, learning that the 5' UTR, CDS, and 3' UTR respectively possess [~]67%, 31%, and 2% per-nucleotide information density in the specification of mammalian TEs. Interpretation of RiboNN revealed that the spatial positioning of low-level di- and tri-nucleotide features (i.e., including codons) largely explain model performance, capturing mechanistic principles such as how ribosomal processivity and tRNA abundance control translational output. RiboNN is predictive of the translational behavior of base-modified therapeutic RNA, and can explain evolutionary selection pressures in human 5' UTRs. Finally, it detects a common language governing mRNA regulatory control and highlights the interconnectedness of mRNA translation, stability, and localization in mammalian organisms.

bioinformatics↗

bioRxiv2024

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH- motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.

molecular biology↗

bioRxiv2024

Cytosolic Ribosomal Protein Haploinsufficiency affects Mitochondrial Morphology and Respiration

The interplay between ribosomal protein composition and mitochondrial function is essential for sustaining energy homeostasis. Precise stoichiometric production of ribosomal proteins is crucial to maximize protein synthesis efficiency while reducing the energy costs to the cell. However, the impact of this balance on mitochondrial ATP generation, morphology and function remains unclear. Particularly, the loss of a single copy ribosomal protein gene is observed in Mendelian disorders like Diamond Blackfan Anemia and is common in somatic tumors, yet the implications of this imbalance on mitochondrial function and energy dynamics are still unclear. In this study, we investigated the impact of haploinsufficiency for four ribosomal protein genes implicated in ribosomopathy disorders (rps-10, rpl-5, rpl-33, rps-23) in Caenorhabditis elegans and corresponding reductions in human lymphoblast cells. Our findings uncover significant, albeit variably penetrant, mitochondrial morphological differences across these mutants, alongside an upregulation of glutathione transferases, and SKN-1 dependent increase in oxidative stress resistance, indicative of increased ROS production. Specifically, loss of a single copy of rps-10 in C. elegans led to decreased mitochondrial activity, characterized by lower energy levels and reduced oxygen consumption. A similar reduction in mitochondrial activity and energy levels was observed in human leukemia cells with a 50% reduction in RPS10 transcript levels. Importantly, we also observed alterations in the translation efficiency of nuclear and mitochondrial electron transport chain components in response to reductions in ribosomal protein genes expression in both C. elegans and human cells. This suggests a conserved mechanism whereby the synthesis of components vital for mitochondrial function are adjusted in the face of compromised ribosomal machinery. Finally, mitochondrial membrane and cytosolic ribosomal components exhibited significant covariation at the RNA and translation efficiency level in lymphoblastoid cells across a diverse group of individuals, emphasizing the interplay between the protein synthesis machinery and mitochondrial energy production. By uncovering the impact of ribosomal protein haploinsufficiency on the translation efficiency of electron transport chain components, mitochondrial physiology, and the adaptive stress responses, we provide evidence for an evolutionarily conserved strategy to safeguard cellular functionality under genetic stress.

genetics↗

bioRxiv2024

RiboGraph: An interactive visualization system for ribosome profiling data at read length resolution

SummaryRibosome profiling is a widely-used technique for measuring ribosome occupancy at nucleotide resolution. However, the need to analyze this data at nucleotide resolution introduces unique challenges in data visualization and analyses. In this study, we introduce RiboGraph, a dedicated visualization tool designed to work with .ribo files, a specialized and efficient format for ribosome occupancy data. Unlike existing solutions that rely on large alignment files and time-consuming preprocessing steps, RiboGraph operates on a purpose designed compact file type and eliminates the need for data preprocessing. This efficiency allows for interactive, real-time visualization at ribosome-protected fragment length resolution. By providing an integrated toolset, RiboGraph empowers researchers to conduct comprehensive visual analysis of ribosome occupancy data. Availability and ImplementationSource code, step-by-step installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling/ribograph. On the same page, we provide test files and a step-by-step tutorial highlighting the key features of RiboGraph.

bioinformatics↗

bioRxiv2024

An inappropriate decline in ribosome levels drives a diverse set of neurodevelopmental disorders

Many neurodevelopmental defects are linked to perturbations in genes involved in housekeeping functions, such as those encoding ribosome biogenesis factors. However, how reductions in ribosome biogenesis can result in tissue and developmental specific defects remains a mystery. Here we describe new allelic variants in the ribosome biogenesis factor AIRIM primarily associated with neurodevelopmental disorders. Using human cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we identify a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, making differentiating cells particularly vulnerable to perturbations in ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing mTOR activity by both genetic and pharmacologic approaches ameliorates the growth and developmental defects associated with intellectual disability linked variants, identifying potential treatment options for specific brain ribosomopathies. This work reveals the cellular and molecular origins of protein synthesis defect-related disorders of human brain development. HighlightsO_LIAIRIM variants reduce ribosome levels specifically in neural progenitor cells. C_LIO_LIInappropriately low ribosome levels cause a transient delay in radial glia fate commitment. C_LIO_LIReduced ribosome levels impair translation of a selected subset of mRNAs. C_LIO_LIGenetic and pharmacologic activation of mTORC1 suppresses AIRIM-linked phenotypes. C_LI

developmental biology↗

bioRxiv2023

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.

molecular biology↗

bioRxiv2023

Integrated multiplexed assays of variant effect reveal cis-regulatory determinants of catechol-O-methyltransferase gene expression

Multiplexed assays of variant effect are powerful methods to profile the consequences of rare variants on gene expression and organismal fitness. Yet, few studies have integrated several multiplexed assays to map variant effects on gene expression in coding sequences. Here, we pioneered a multiplexed assay based on polysome profiling to measure variant effects on translation at scale, uncovering single-nucleotide variants that increase and decrease ribosome load. By combining high-throughput ribosome load data with multiplexed mRNA and protein abundance readouts, we mapped the cis-regulatory landscape of thousands of catechol-O-methyltransferase (COMT) variants from RNA to protein and found numerous coding variants that alter COMT expression. Finally, we trained machine learning models to map signatures of variant effects on COMT gene expression and uncovered both directional and divergent impacts across expression layers. Our analyses reveal expression phenotypes for thousands of variants in COMT and highlight variant effects on both single and multiple layers of expression. Our findings prompt future studies that integrate several multiplexed assays for the readout of gene expression.

genomics↗

bioRxiv2022

satmut_utils: a simulation and variant calling package for multiplexed assays of variant effect

BackgroundThe impact of thousands of individual genetic variants on molecular phenotypes for disease-relevant genes remains unknown. Multiplexed assays for variant effect (MAVEs) are highly scalable methods to annotate the relevant variants. However, current software methods for analyzing MAVEs lack standardized annotation, can require cumbersome configuration, and do not easily scale to large target regions. ResultsHere, we present satmut_utils as a flexible solution for 1) simulation of saturation mutagenesis data; and 2) quantification of variants across four orders of magnitude from multiplexed assay data. Improvements of satmut_utils over existing solutions include support for multiple experimental strategies, unique molecular identifier-based consensus deduplication, and machine learning-based error correction. We developed a rigorous simulation workflow to validate the performance of satmut_utils and carried out the first benchmarking of existing software for variant calling. Finally, we used satmut_utils to determine the mRNA abundance of thousands of coding variants in cystathionine beta-synthase (CBS) by two library preparation methods. We identified an association between variants near chemical cofactor binding sites and decreased mRNA abundance. We also found a correlation between codon optimality and the magnitude of variant effects, emphasizing the potential of single-nucleotide variants to alter mRNA abundance. Conclusionssatmut_utils enables high-performance analysis of saturation mutagenesis data, achieves unprecedented specificity through novel error correction approaches, and reveals the capability of single-codon variants to alter mRNA abundance in native coding sequences.

genomics↗