Biology subjects

Sendoel, A.

Publications and source records attributed to Sendoel, A..

9 recordsLinked to original sources

bioRxiv2026

rhapsodist: a reproducible Snakemake workflow for BD~Rhapsody single-cell RNA-seq data

BD Rhapsody is a widely used single-cell RNA-seq (scRNA-seq) platform to profile gene expression. Based on barcoded beads, processing BD Rhapsody experiments is demanding because of the multiple barcode versions and layouts, which makes it difficult to locate barcodes by scanning reads positionally. We introduce rhapsodist, a reproducible and scalable Snakemake workflow to process BD Rhapsody whole transcriptome analysis (WTA) data from raw FASTQ reads. As a result, rhapsodist generates count tables in a HDF5-backed SingleCellExperiment format. It supports all generations of BD Rhapsody beads, regardless of the barcode allowlist version and the presence of a variable-length diversity inset. rhapsodist orchestrates STARsolo, kallisto/bustools, salmon/alevin, and the official BD Rhapsody CWL pipeline, enabling direct cross-aligner comparison. Optionally, sample tag demultiplexing, read downsampling, tunable linker-mismatch tolerance, and per-sample reports are provided. We showcase a remarkable consistency across aligner results, and highlight their differences in performance (e.g., quantification, clustering, or speed). On simulated data all four aligners recovered true barcodes with perfect precision and recall and equivalent UMI counts. On HeLa cells (a cell line; enhanced beads), both per-cell correlations across aligners and pseudobulk correlations were remarkably high on every comparison (Pearson r of 0.89-0.96 and 0.94-0.98 per cell and pseudobulk, respectively). On a mouse epidermis dataset with a more complex transcriptional profile (e.g., with celltypes; on legacy beads), cell type pseudobulk correlations showed a similar consistency (Pearson r 0.92-0.98), pointing to calling robustness. As expected, pseudoaligners (kallisto/bustools and salmon/alevin) were best performers speed-wise, and BDs workflow recovered a higher number of barcodes and UMIs. rhapsodist is available at https://github.com/imallona/rhapsodist under GPLv3, tried on Linux with conda, and includes unit and integration tests.

bioinformatics↗

bioRxiv2025

Regulation of mRNA polyadenylation governs mammalian body plan formation in gastruloids

The establishment of the body plan during gastrulation represents a hallmark of animal life. It emerges from the interplay of gene-regulatory programs and positional cues, yet how these signals are integrated post-transcriptionally remains largely unexplored. Here, we combine the scalability of mouse gastruloids with a single-cell CRISPR screening platform to functionally dissect germ layer specification at single-cell transcriptomic resolution. Focusing on post-transcriptional regulation, we systematically map drivers of mesodermal and endodermal fate and identify the deadenylase Cnot8. Loss of Cnot8 leads to widespread poly(A) tail elongation and transcript stabilization, shifting mesoderm differentiation toward ectopic notochord fate, thereby profoundly impacting axial patterning. Collectively, our findings identify mRNA deadenylation as a fundamental mechanism linking cellular identity with morphogenetic signaling during mammalian body plan formation.

developmental biology↗

bioRxiv2025

The alternative initiation factor eIF2A regulates 40S subunit turnover in ribosome-associated quality control

The noncanonical translation initiation factor eIF2A plays critical roles in diverse cellular processes, including the integrated stress response, neurodegeneration and tumorigenesis. However, the precise molecular mechanism underlying eIF2As function remains poorly understood. Here, we exploit a TurboID-based proximity labeling combined with mass spectrometry to systematically map the interactome of eIF2A during homeostasis and stress. Combining polysome gradients with TurboID, we zoom into the interactions of eIF2A with the 40S small ribosomal subunit and map the eIF2A binding site close to the mRNA entry channel. We identify a network of interactors that link eIF2A to ribosome-associated quality control, including its strong interaction with G3BP1-USP10 complexes as well as RPS2 and RPS3. In the absence of eIF2A, RPS2 and RPS3 ubiquitination is diminished specifically upon ribosome stalling. 40S-specific footprinting in eIF2A knockout cells shows minimal changes in 5UTR occupancy, consistent with a limited role for eIF2A in translation initiation. Using dynamic SILAC mass spectrometry, we characterize the novel function of eIF2A in ribosome-associated quality control and show that eIF2A antagonizes USP10-dependent rescue of 40S ribosomes, resulting in altered turnover of 40S subunits upon cellular stress. Collectively, our study identifies a previously unknown link between eIF2A and ribosome-associated quality control, implies that eIF2A promotes translation fidelity by tuning 40S ribosome rescue under stress and warrants further investigations into the role of ribosome-associated quality control in tumorigenesis.

molecular biology↗

bioRxiv2025

In vivo single-cell CRISPR screening for microproteins identifies a critical ribosomal component

The dark proteome includes a rapidly expanding catalog of microproteins with unknown functions that have been historically ignored in genome annotations. Here, we exploit an in vivo single-cell CRISPR screening strategy in the mouse epidermis to systematically investigate the tissue-wide function of microproteins. We document the global and cell-type-specific roles of microproteins during epidermal development and homeostasis at single-cell transcriptomic resolution. Focusing on select candidates, we identify a novel microprotein on Gm10076, identical to the ribosomal intersubunit bridge protein RPL41, whose perturbation strongly impairs proliferation and protein synthesis. Employing ribosome profiling and RNA sequencing, we show that Gm10076 perturbation profoundly reshapes the translational landscape. Contrary to its prior classification as nonessential, we find that the ribosomal protein RPL41 is essential for cellular proliferation, warranting further investigation into its role as an intersubunit bridge in the translational machinery. Together, our study comprehensively charts the tissue-wide functional landscape of the dark proteome, uncovers a second Rpl41 gene critical for ribosome function and establishes a basis for exploring the impact of microproteins on disease pathogenesis.

genetics↗

bioRxiv2024

In vivo single-cell ribosome profiling reveals cell-type-specific translational programs during aging

Somatic stem cells are characterized by their low overall protein synthesis rates, a feature implicated in driving their stemness. However, how aging reshapes the translational landscape of stem cells and how these changes impact their regenerative capacity remains poorly understood. Here, we present an in vivo single-cell ribosome profiling strategy to monitor tissue-wide translational landscapes of the young and aged mouse epidermis. By implementing ribosomal elongation-inhibited cell isolation and switching to RNase I ribonuclease for generating ribosomal footprints, we expand the applicability of single-cell ribosome profiling to in vivo systems and facilitate the evaluation of triplet periodicity, a hallmark of high-quality ribosome profiling data. Leveraging this strategy and integrating ribosome profiling with single-cell RNA sequencing, we document the in vivo translational landscapes of the major epidermal cell types, outline cell-type-specific translational efficiencies and capture heterogeneity in differentiation commitment within stem cell populations. Notably, we identify a pronounced translational reprogramming of AP-1 subunits specifically in aged epidermal stem cells, with functional consequences for keratinocyte behavior. Our study illustrates the power of in vivo single-cell ribosome profiling to map cell-type-specific translational programs and offers a scalable strategy for tissue-wide interrogation of translational landscapes at single-cell resolution.

genomics↗

bioRxiv2023

In vivo single-cell CRISPR uncovers distinct TNF-alphaprograms in clonal expansion and tumorigenesis

The tumor evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions occur frequently in human epithelia and can remodel almost entire tissues, the mechanisms behind why only a small number of clones transform into malignant tumors remain enigmatic. Here, we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing, guide capture and spatial transcriptomics to longitudinally monitor cell type-specific clonal expansions, document their underlying gene programs and contrast clonal expansions from tumor initiation. We uncover a TNF- signaling module that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF- signaling module is downregulated, and instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF- gene program. By analyzing clonally expanded perturbations and their frequency in tumors, we demonstrate that the autocrine TNF- gene program is associated with epithelial-mesenchymal transition (EMT) and is preexistent in a subpopulation of expanded epidermal stem cells, contributing to the predisposition for tumor initiation. Finally, we provide in vivo evidence that the epithelial TNF- gene program is sufficient to mediate invasive properties of epidermal stem cells and show that the TNF- signature correlates with shorter overall survival in human squamous cell carcinoma patients. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues and unveils distinct TNF- programs in tumor evolution. Understanding the biology of clonal expansions in phenotypically normal epithelia and the mechanisms governing their transformation will guide the development of novel strategies for early cancer detection and therapy.

cancer biology↗

bioRxiv2022

In vivo CRISPR screens reveal SCAF1 and USP15 as novel drivers of pancreatic cancer

Functionally characterizing the genetic alterations that drive pancreatic cancer progression is a prerequisite for Precision Medicine. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to assess the transforming potential of 125 recurrently mutated long-tail pancreatic cancer genes, which revealed USP15 and SCAF1 as novel and potent Pancreatic ductal adenocarcinoma PDAC tumor suppressors, with USP15 functioning in a haplo-insufficient manner. Mechanistically, we found that loss of USP15 leads to reduced inflammatory responses associated with TNF, TGF-{beta} and IL6 signaling and sensitizes pancreatic cancer cells to PARP inhibition and gemcitabine. Similarly, genetic ablation of SCAF1 reduced inflammatory responses linked to TNF, TGF-{beta} and mTOR signaling and increased sensitivity to PARP inhibition. Furthermore, we identified that loss of SCAF1 resulted in the formation of a truncated inactive USP15 isoform at the expense of full length USP15, functionally coupling SACF1 and USP15. Notably, USP15 and SCAF1 mutations or copy number losses are observed in 31% of PDAC patients. Together, our results demonstrate the utility of in vivo CRISPR to integrate human cancer genomics with mouse modeling to delineate novel cancer driver genes USP15 and SCAF1 such as with potential prognostic and therapeutic implications.

cancer biology↗

bioRxiv2022

The integrated stress response remodels the microtubule organizing center to clear unfolded proteins following proteotoxic stress

When cells encounter stressful situations, they activate the integrated stress response (ISR), which limits total protein synthesis and redirects translation to proteins that help the cells to cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely the S51 phosphorylation site of eukaryotic translation initiation factor eIF2 and genetically replaced eIF2 with eIF2-S51A in squamous cell carcinoma (SCC) stem cells. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during stress, the ISR also functions in promoting cellular recovery once the stress has subsided. This finding exposes a vulnerability to SCC stem cells that could be exploited therapeutically.

cell biology↗

bioRxiv2021

Monitoring the 5`UTR landscape reveals 5`terminal oligopyrimidine (TOP) motif switches to drive translational efficiencies

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here we use long-read sequencing to document the 5and 3untranslated region (UTR) isoform landscape of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. These 5UTR isoforms with identical coding sequences either include or exclude 5terminal oligopyrimidine (TOP) motifs and result in vastly altered translational efficiencies of the mRNA. Our findings suggest that switching between TOP and non-TOP motif-containing 5UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5UTR sequence.

cancer biology↗