Biology subjects

Leidel, S.

Publications and source records attributed to Leidel, S..

5 recordsLinked to original sources

bioRxiv2026

Harmonized nucleoside mass spectrometry enables reproducible cross-platform RNA modification quantification

RNA modification analysis by LC-MS/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside quantification across quadrupole, time-of-flight, and orbitrap-based LC-MS platforms employing distinct acquisition strategies. While chromatographic separation was highly reproducible across systems, nucleoside-specific MS response behavior differed substantially between platforms and limited direct comparability of relative signal intensities. These response differences varied across analytes and concentration ranges, demonstrating that harmonized chromatography alone is insufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform-and method-dependent response and improved agreement for most evaluated modifications. External calibration improved agreement between qTOF and Orbitrap workflows for a subset of modifications but did not fully resolve residual intersystem differences. Based on these findings, we establish benchmark-derived recommendations for harmonized relative and absolute RNA modification quantification, including guidance for calibration design, quality control, and data reporting. Together, this work provides a methodological framework for reproducible nucleoside LC-MS/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/739095v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@6e4e98org.highwire.dtl.DTLVardef@1ea13beorg.highwire.dtl.DTLVardef@b65858org.highwire.dtl.DTLVardef@1623337_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Loss of host factor-mediated m6Am methylation of the viral RNA cap impairs SARS CoV-2 replication

Eukaryotic mRNAs are co-transcriptionally capped at the 5' end with a methylated m7G moiety (cap0)1, which in higher eukaryotes is further methylated on the ribose (Nm) of the transcription start site (TSS) nucleotide to create the cap1 structure (m7GpppNm). Coronaviruses that replicate in the cytoplasm encode their own capping enzymes to acquire this cap1 structure which facilitates translation and shields them from the host innate immune system2-5. Here we report the identification of an additional N6-methyladenosine (m6A) methylation on the 5' cap (m7Gpppm6Am) of the human coronavirus SARS-CoV-2 RNA. It is catalysed by the host m6A methylase PCIF16-9 following capping by virus-encoded non-structural protein NSP 14 and NSP1610. Human cell cultures lacking PCIF1 accumulate reduced levels of the viral RNA and support reduced viral replication. Furthermore, Pcif1 mutant mice infected with SARS CoV-2 display milder symptoms. We identify the host RNA methyltransferase PCIF1 as a critical ally of SARS CoV-2 for viral replication.

molecular biology↗

bioRxiv2025

Pus7 mutation links tRNA dysregulation to aggressive behavior through activation of the integrated stress response and glycolytic reprogramming

Pseudouridine ({Psi}) is a prevalent RNA modification found in multiple RNA species. It is deposited by {Psi} synthases (Pus) and it can stabilize RNA structures. Patients carrying alterations in the Pus7 gene suffer from developmental delay, intellectual disability, microcephaly, hyperactivity and increased aggression levels. Here we show that the Pus7 mutation in human patient cells and in a Drosophila model is associated with a specific decrease of tRNA:Aspartate (tRNA-Asp) levels, which leads to slow decoding at Aspartate codons. This in turn activates the integrated stress response and induces a metabolic shift towards increased glycolysis and reduced mitochondrial respiration. Elevating tRNA-Asp expression, inhibiting the integrated stress response or dampening the glycolytic pathway is sufficient to rescue the aggressiveness phenotype, demonstrating the involvement of the tRNA-Asp-ISR-glycolysis axis in this behavior. Together our data provide new insights into the molecular defects associated with the loss of Pus7 and suggest potential new avenues for therapeutic treatment.

genetics↗

bioRxiv2022

A rapid protocol for ribosome profiling of low input samples

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected from nucleolytic digestion by ribosomes. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of RNA. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

systems biology↗

bioRxiv2017

Attomol-level quantification of chemically modified ribonucleosides enabled by capillary porous graphitic carbon columns in nano LC-MS

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited the development of methods for reliable detection and accurate quantification of these modifications. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials as stationary phases for reversed-phase separation of ribonucleosides and found that both PGC matrices have excellent retention and separation characteristics, as well as the capability to separate structural isomers. Using PGC matrices in nLC-MS yielded excellent signal-to-noise ratios in a detection range spanning up to six orders of magnitude, allowing for the analysis of individual ribonucleosides down to attomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary PGC columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of chemically modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

biochemistry↗