Biology subjects

Verniers, K.

Publications and source records attributed to Verniers, K..

8 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↗

bioRxiv2024

Intact messenger RNA exists in human blood plasma and urine, and their purified macromolecular compartments

It is generally assumed that extracellular long RNA molecules in biofluids are fragmented. Few studies have indirectly hinted at the existence of possibly functional, intact long RNA transcripts. In search for such RNA molecules, we developed a long-read full transcript sequencing workflow for low-input and low-quality samples. We applied our method to human blood plasma, urine, and their isolated macromolecular compartments, in parallel with total RNA sequencing. This approach enabled us to find intact messenger RNA molecules in human biofluids and macromolecular compartments. We showed that the full-length transcriptome of human urine and blood plasma differs, but we also reveal intact messenger RNA molecules shared between biofluids. In addition, we show that these intact molecules are differentially distributed over fractionated macromolecular compartments. This study provides a foundation for future extracellular RNA studies to elucidate the human biofluid full-length transcriptome.

molecular biology↗

bioRxiv2022

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed by computational detection tools. During the last decade, a plethora of such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools were used and detected over 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were empirically validated using three orthogonal methods. Generally, tool-specific precision values are high and similar (median of 98.8%, 96.3%, and 95.5% for qPCR, RNase R, and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant tool differentiators. Furthermore, we demonstrate the complementarity of tools through the increase in detection sensitivity by considering the union of highly-precise tool combinations while keeping the number of false discoveries low. Finally, based on the benchmarking results, recommendations are put forward for circRNA detection and validation.

genomics↗

bioRxiv2022

Blocking unwanted sequences by target specific high-affinity binding oligonucleotides during transcriptome library preparation

RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3 end sequencing and long-read sequencing, and MALAT1 in single-cell 3 end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Furthermore, our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

molecular biology↗

bioRxiv2021

A 3'-end capture sequencing method for high-throughput targeted gene expression profiling

Molecular phenotyping through shallow 3-end RNA-sequencing workflows is increasingly applied in the context of large-scale chemical or genetic perturbation screens to study disease biology or support drug discovery. While these workflows enable accurate quantification of the most abundant genes, they are less effective for applications that require expression profiling of low abundant transcripts, like long non-coding RNAs (lncRNAs), or selected gene panels. To tackle these issues, we describe a workflow combining 3-end library preparation with 3-end hybrid capture probes and shallow RNA-sequencing for cost-effective, targeted quantification of subsets of (low abundant) genes across hundreds to thousands of samples. To assess the performance of the method, we designed a capture probe set for more than 100 mRNA and lncRNA target genes and applied the workflow to a cohort of 360 samples. When compared to standard 3-end RNA-sequencing, 3-end capture sequencing resulted in a more than 100-fold enrichment of target gene abundance while conserving relative inter-gene and inter-sample abundances. 3-end RNA capture sequencing enables accurate targeted gene expression profiling at extremely shallow sequencing depth.

genomics↗

bioRxiv2021

Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

The use of blood-based extracellular RNA (cell-free RNA; exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing, and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated ten blood collection tubes, three time intervals between blood draw and downstream processing, and eight RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics on deep transcriptome profiling of both small and messenger RNA from healthy donors plasma or serum was assessed for each pre-analytical variable separately and for interactions between a selected set of pre-analytics, resulting in 456 extracellular transcriptomes. Making use of 189 synthetic spike-in RNAs, the processing and analysis workflow was controlled. When comparing blood collection tubes, so-called preservation tubes do not stabilize exRNA well, and result in variable RNA concentration and sensitivity (i.e., the number of detected RNAs) over time, as well as compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity, and demonstrate interactions between specific blood collection tubes, purification kits and time intervals. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for a given experiment. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs), representing paramount groundwork for future exRNA-based precision medicine applications.

genomics↗

bioRxiv2019

Charting extracellular transcriptomes in The Human Biofluid RNA Atlas

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on plasma or serum, other biofluids may contain more informative RNA molecules, depending on the type of disease. Here, we present an unprecedented atlas of messenger, circular and small RNA transcriptomes of a comprehensive collection of 20 different human biofluids. By means of synthetic spike-in controls, we compared RNA content across biofluids, revealing a more than 10 000-fold difference in RNA concentration. The circular RNA fraction is increased in nearly all biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. In addition, a subset of biofluids, including stool, sweat, saliva and sputum, contains high levels of bacterial RNAs. Our atlas enables a more informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Taken together, our results reveal novel insights in the RNA content of human biofluids and may serve as a valuable resource for future biomarker studies. All spike-normalized data is publicly available in the R2 web portal and serve as a basis to further explore the RNA content in biofluids.

genomics↗

bioRxiv2019

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

molecular biology↗