Biology subjects

Rombaut, D.

Publications and source records attributed to Rombaut, D..

6 recordsLinked to original sources

bioRxiv2025

Stress attenuation by the adrenergic-specific lncRNA NESPR prevents cell death in neuroblastoma cells

Neuroblastoma is a pediatric cancer of the sympathetic nervous system characterized by heterogeneous cell states that mirror normal differentiation trajectories. Each state is governed by a core regulatory transcriptional circuitry that reinforces cell identity through an autoregulatory feedforward loop. We identified the long non-coding RNA NESPR as specifically expressed in adrenergic neuroblastoma cells. NESPR expression correlates with high-risk neuroblastoma clinical parameters and poor patient survival. NESPR is located within an insulated gene neighborhood alongside PHOX2B, a master transcription factor of the adrenergic identity. NESPR depletion reduced cell proliferation and increased caspase activity in neuroblastoma cell lines, and NESPR knockout in a neuroblastoma zebrafish model led to reduced tumor penetrance. Subcellular localization revealed NESPR to be a cytosolic long non-coding RNA, suggesting a trans-regulatory function. RNA-sequencing following NESPR depletion revealed a shift from an adrenergic to a mesenchymal cell state, due to proteotoxic stress-induced molecular reprogramming. These findings suggest NESPR as a regulator of neuroblastoma cell identity with potential therapeutic opportunities in high-risk neuroblastoma cases.

genetics↗

bioRxiv2023

Continual improvement of multiplex mutagenesis in Arabidopsis

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side-by-side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported and can still be optimized. We genotyped more than 6,000 Arabidopsis T2 plants to test seven promoters and eleven NLS architectures across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We find that the RPS5A promoter and double-BP NLS architecture were individually the most efficient components. When combined, 99% of T2 plant contained at least one knockout mutation and 84% contained 4-7-plex knock-outs. These optimizations will be useful to generate higher-order knockouts in the germline of Arabidopsis and likely be applicable to other CRISPR systems as well.

synthetic biology↗

bioRxiv2020

Comprehensive benchmarking of single cell RNA sequencing technologies for characterizing cellular perturbation

Technological advances in transcriptome sequencing of single cells continues to provide an unprecedented view on tissue composition and cellular heterogeneity. While several studies have compared different single cell RNA-seq methods with respect to data quality and their ability to distinguish cell subpopulations, none of these studies investigated the heterogeneity of the cellular transcriptional response upon a chemical perturbation. In this study, we evaluated the transcriptional response of NGP neuroblastoma cells upon nutlin-3 treatment using the C1, ddSeq and Chromium single cell systems. These devices and library preparation methods are representative for the wide variety of platforms, ranging from microfluid chips to droplet-based systems and from full transcript sequencing to 3-prime end sequencing. In parallel, we used bulk RNA-seq for molecular characterization of the transcriptional response. Two complementary metrics to evaluate performance were applied: the first is the number and identity of differentially expressed genes as defined in consensus by two statistical models, and the second is the enrichment analysis of biological signals. Where relevant, to make the data more comparable, we downsampled sequencing library size, selected cell subpopulations based on specific RNA abundance features, or created pseudobulk samples. While the C1 detects the highest number of genes per cell and better resembles bulk RNA-seq, the Chromium identifies most differentially expressed genes, albeit still substantially fewer than bulk RNA-seq. Gene set enrichment analyses reveals that detection of a limited set of the most abundant genes in single cell RNA-seq experiments is sufficient for molecular phenotyping. Finally, single cell RNA-seq reveals a heterogeneous response of NGP neuroblastoma cells upon nutlin-3 treatment, revealing putative late-responder or resistant cells, both undetected in bulk RNA-seq experiments.

molecular biology↗

bioRxiv2020

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Advanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Here we reveal, in the plant model Arabidopsis thaliana, that the PIN7 gene, which encodes a polarly localized transporter for the phytohormone auxin, produces two evolutionary-conserved transcripts. These isoforms PIN7a and PIN7b, differing in a 4 amino acid motif, are present at nearly equal levels in most cells. Although both variants do not differ in the subcellular localization and transport auxin with similar capacity, they closely associate and mutually influence their stability within the plasma membrane. Phenotypic complementation tests reveal that the functional contribution of PIN7b per se is minor but it markedly attenuates the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. These results establish alternative splicing of the PIN family as a conserved, functionally-relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.

plant biology↗

bioRxiv2018

SMARTer single cell total RNA sequencing

Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3 end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.

genomics↗

bioRxiv2018

Genomic from Plasmodium falciparum field isolates from Benin allows the identification of PfEMP1 variants by LC-MS/MS at the patient’s level

PfEMP1 are the major protein family from parasitic origin involves in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability in endless and current protein repository do not reflect the immense diversity of the sequences of PfEMP1 proteins. The aim of our study was to identify the different PfEMP1 variants expressed within a patient sample by mass spectrometry. We performed a proteogenomic approach to decipher at the patients level PfEMP1 expression in different clinical settings: cerebral malaria, severe anemia and uncomplicated malaria. The combination of whole genome sequencing approach, RNAsequencing, and mass spectrometry proteomic analysis allowed to attribute PfEMP1 sequences to each sample and classify the relative expression level of PfEMP1 proteins within each sample. We predicted PfEMP1 structures using the newly identified protein sequences. We confirmed the involvement of DBL{beta} in malaria pathogenesis and observed that CIDR domains linked to ICAM-1 binding DBL{beta} domains displayed EPCR binding structure.

microbiology↗