Biology subjects

Verwilt, J.

Publications and source records attributed to Verwilt, J..

4 recordsLinked to original sources

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↗

bioRxiv2024

Deletion of an sRNA primes development in a multicellular bacterium

ABSTRACTSmall non-coding RNAs (sRNAs) are essential in regulating gene expression during many biological processes. The myxobacteria gene pxr encodes an sRNA known to block fruiting-body development, an aggregative multicellular process triggered by starvation. Deletion of pxr allows Myxococcus xanthus cells to develop in the presence of nutrients. However, potential Pxr binding targets and most genes regulated by Pxr remain unknown. Here, we found that the absence of pxr expression dramatically alters the temporal dynamics of development, thus suggesting an important new role of this sRNA in myxobacterial ecology. We transcriptionally profiled vegetative cells of M. xanthus strains possessing vs lacking pxr and found that over half of the genes impacted by pxr deletion during growth are linked to development, including known and potentially novel critical regulators. Many other genes are associated with general metabolic processes, which Pxr regulates positively. Our study discovers new phenotypic effects of Pxr regulation of likely ecological importance, identifies the suite of genes this sRNA controls during vegetative growth, reveals a previously unknown developmental regulator and provides new insights into the early molecular regulation of myxobacterial development.

molecular biology↗

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↗

bioRxiv2020

When DNA gets in the way in RNA-seq experiments, a sequel

Using a newly developed method dubbed SILVER-Seq--enabling extracellular RNA sequencing (exRNA-seq) directly from a small volume of human serum or plasma-- Yan et al. recently reported in Current Biology a potential exRNA biomarker for the early diagnosis of Alzheimers disease [1]. After the publication of the initial paper describing the SILVER-Seq method [2], we reported our concern regarding potential DNA contamination in their datasets [3]. Although the authors replied they were able to successfully treat RNA samples with DNase to avoid such contamination, they did not address our observations of the majority of reads without evidence of being derived from RNA, nor documented verified absence of DNA after DNase treatment [4]. To assess whether the newly data generated may suffer from DNA contamination, we downloaded the publicly available sequencing data and evaluated two quality control metrics (i.e., fraction of exonic and splice reads), which were not reported in the paper. We found that both quality metrics were much lower than expected for RNA-seq data (6.28% exonic and 0.478% splice reads), in line with our previous findings on the first SILVER-Seq paper. These observations suggest the data and results presented by Yan et al. are affected by DNA contamination, an issue that may be inherent to the SILVER-Seq technology.

molecular biology↗