Biology subjects

Wolff, P.

Publications and source records attributed to Wolff, P..

12 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

Human 28S rRNA analysed by state-of-the-art oligonucleotide mass spectrometry: benchmarking current capabilities and a call to action for MS-Seq

Oligonucleotide mass spectrometry (MS-Seq) is emerging as a powerful approach for sequence-resolved RNA modification analysis, yet the field lacks standards for experimental workflows, data analysis and reporting. To assess current capabilities, the Human RNome Project Consortium conducted a cross-platform benchmarking study using a common RNA sample. A partial RNase T1 digest of human 28S rRNA was distributed to participating laboratories and analysed using existing LC-MS/MS workflows spanning different chromatographic strategies and mass spectrometers. To enable direct comparison, datasets were analysed using a harmonized NucleicAcidSearchEngine (NASE) workflow. Despite substantial methodological differences, laboratories recovered highly overlapping oligonucleotide sets and generated similar sequence coverage maps with a global coverage of 54.16%, demonstrating reproducible sequence information across platforms under standardized sample and analysis conditions. The benchmark further revealed incomplete sequence coverage, platform-specific differences in data architecture and increased assignment ambiguity during dynamic modification searches. Together with the community consensus developed during the HRPC workshop, these findings define priorities for the field, including improved sensitivity, standardized data analysis and reporting, community repositories, and robust bioinformatic workflows for confident de novo RNA modification discovery. This study provides an experimental benchmark and roadmap toward routine MS-based mapping of the human RNome. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/739151v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1fb1b9borg.highwire.dtl.DTLVardef@d172a5org.highwire.dtl.DTLVardef@bdc185org.highwire.dtl.DTLVardef@1ec1cc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Longitudinal blood DNA methylation profiling reveals disrupted immune-epigenetic adaptation and candidate stress related loci in postpartum depression

Postpartum depression (PPD) arises during a period of profound endocrine and immune reorganisation, yet it is unclear whether women who develop PPD show distinct trajectories of immune-related DNA methylation compared to euthymic mothers. In a longitudinal cohort, women with PPD (n = 17) and healthy postpartum controls (n = 24) were followed from birth to 12 weeks postpartum, with repeated assessment of depressive symptoms and perceived stress and whole-blood sampling at 2-3 days (T0) and 12 weeks (T4) for Infinium MethylationEPIC array profiling. Healthy postpartum women showed a widespread gain in DNA methylation from T0 to T4 with strong enrichment of genes involved in neutrophil activation, chemokine signalling and interleukin-1 production, consistent with a normative immune-epigenetic down-tuning after childbirth. Women with PPD also exhibited immune-related changes, but with fewer differentially methylated CpGs and increased variance at sites that were stably hypermethylated in controls, indicating an attenuated and more heterogeneous epigenetic response. Although no CpG reached epigenome-wide significance in direct case-control contrasts, longitudinal consistency analyses highlighted a small set of CpGs with reproducible PPD-associated hypermethylation in stress- and signalling-related genes, including FKBP5 and AVP, suggesting that disrupted immune-epigenetic adaptation and altered regulation at these loci may contribute to postpartum vulnerability.

molecular biology↗

bioRxiv2025

Complete post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

Transfer RNAs play a critical role in protein synthesis by matching mRNA codons to their corresponding amino acids. Their post-transcriptional modifications, shaping structure, stability, and codon decoding, are now recognized as key regulators of translation and cellular adaptation, including in bacterial pathogens. Here, we provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including the non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of [m2A]37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and a dedicated pseudouridine synthase responsible for [{Psi}]32 formation. Besides, heterogenous modification patterns were observed in tRNALeu(UAA) and tRNALys(CAA), highlighting a complex interplay in anticodon hypermodification. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of the decoding properties of the reduced S. aureus tRNA set, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework for future research aimed at dissecting the role of specific tRNA modifications in S. aureus physiology and pathogenesis.

molecular biology↗

bioRxiv2025

A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

Proteins traditionally confined to a single cellular compartment are increasingly recognized to exert non-canonical functions in alternative domains. The DNA methyltransferase 1 (DNMT1), classically defined as the maintenance methyltransferase that preserves DNA methylation patterns during replication, exemplifies this versatility. Beyond its canonical role, DNMT1 is highly expressed in the developing and adult brain, where it contributes to transcriptional regulation in postmitotic neurons. Notably, cytoplasmic DNMT1 localization has been observed in neural cells, and emerging evidence links DNMT1 to mitochondrial function with implications for neurodegenerative disease, whereby the underlying functional mechanisms remain to be fully elucidated. Here, we identify a previously unrecognized cytosolic function of DNMT1 in developing cortical excitatory neurons. Through genetic perturbation, proteomics, and high-resolution imaging, we show that DNMT1 regulates dendritic and axonal branching independently of its catalytic activity and nuclear localization. Instead, DNMT1 operates as a cytosolic scaffold interacting with the polarity regulator DOCK7 to modulate Rac1-STMN1 signaling, microtubule dynamics, and organelle trafficking. These findings expand the conceptual framework of DNMT1 from a genome guardian to a dual-compartment regulator that coordinates cytoskeletal remodeling and mitochondrial positioning. Beyond advancing our understanding of neuronal morphogenesis, this work provides mechanistic insight into how DNMT1 mutations may lead to neurodegenerative diseases.

neuroscience↗

bioRxiv2024

DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

The fine-tuned establishment of neuronal circuits during the formation of the cerebral cortex is pivotal for its functionality. Developmental abnormalities affecting the composition of cortical circuits, which consist of excitatory neurons and inhibitory cortical interneurons (cINs), are linked to a spectrum of neuropsychiatric disorders. Excitatory neurons originate in cortical proliferative zones, while inhibitory interneurons migrate from discrete domains of the basal telencephalon into the cortex. This migration is intricately governed by intrinsic genetic programs and extrinsic cues. Our current study reveals the role of the DNA methyltransferase 1 (DNMT1) in regulating the expression of key genes implicated in mouse cIN development and in guiding the migration of somatostatin (SST)-expressing interneurons at postmitotic level within the developing cortex. Dnmt1 deletion causes SST+ cINs to exit prematurely from the superficial migratory stream. In addition to the perturbed migration pattern and altered gene expression signatures, Dnmt1-deficient SST+ cINs had a discernible non-cell autonomous effect on cortical progenitors, which culminated in nuanced alterations of layer thicknesses in the adult cortex. Our study uncovers that DNMT1 governs the migration of SST+ cINs and through this, their instructive role in sculpting the intricate cortical layer architecture by signaling to cortical progenitors, with pronounced effects on neuronal network function.

neuroscience↗

bioRxiv2023

Structure of the human 80S ribosome at 1.9 A resolution - the molecular role of chemical modifications and ions in RNA

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. We present the 1.9 [A] resolution cryo-EM structure of the 80S human ribosome resolving numerous new rRNA modifications and functionally important ions such as Zn2+, K+ and Mg2+ including their associated individual water molecules. 2-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry resulting in a complete investigation of the > 230 sites many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerisation turns out to be a key mechanism for U-A base pair stabilisation in RNA in general. The structural environment of chemical modifications & ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy context and human diseases.

biochemistry↗

bioRxiv2023

RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in Staphylococcus aureus

rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analysis of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general as well as species-specific roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in E. coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.

molecular biology↗

bioRxiv2023

Antisense transcription and PRC2 repression function in parallel during vernalization

Non-coding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription interfaces with Polycomb Repressive Complex 2 silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). Through genetic, chromatin, and computational analyses, we show that FLC is silenced through pathways that function with different dynamics: an antisense transcription-mediated pathway capable of fast response; and in parallel a slow Polycomb Repressive Complex 2 (PRC2) switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the antisense and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates slowly due to reduced growth at low temperatures. The parallel activities of the regulatory steps, which we encapsulate in a mathematical model, creates a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC. SignificanceThe role of non-coding transcription in establishing and maintaining chromatin states is controversial, mainly because of extensive feedbacks complicating analysis of the relationship between co-transcriptional processing, chromatin state and transcription. This controversy has extended to the role of antisense transcription in the Polycomb-mediated epigenetic silencing of Arabidopsis FLC, a key step in the process of vernalization. Here, we show that antisense transcription and PRC2 silence FLC in parallel pathways that are affected by growth dynamics and temperature fluctuations. These features explain the varied importance of antisense transcription in cold-induced FLC epigenetic silencing seen in various studies using different environmental and growth conditions. The parallel repressive inputs and extensive feedbacks make the mechanism counter-intuitive but provide great flexibility to the plant.

molecular biology↗

bioRxiv2021

VAL1 as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC

Polycomb (PcG) silencing is crucial for development across eukaryotes, but how PcG targets are regulated is still incompletely understood. The slow timescale of cold-induced PcG silencing at Arabidopsis thaliana FLOWERING LOCUS C (FLC) makes it an excellent system to dissect this mechanism. Binding of the DNA binding protein VAL1 to an FLC intronic RY motif within the PcG nucleation region is an early step in the silencing process. VAL1 interacts with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and POLYCOMB REPRESSIVE COMPLEX 1 (PRC1). Here, we show that ASAP and PRC1 function as co-repressors that quantitatively regulate FLC transcription. Upon the shift to cold PRC1-mediated H2Aub accumulates only at the nucleation region, is transiently maintained after transfer back to warm, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2K27me3 thus provides long-term epigenetic silencing, whereas H2Aub is a transient repression signal. Overall, our work highlights how a DNA sequence-specific binding protein can act as an assembly platform co-ordinating the co-transcriptional repression and chromatin regulation necessary for Polycomb silencing.

molecular biology↗

bioRxiv2017

Paternal easiRNAs regulate parental genome dosage in Arabidopsis

The regulation of parental genome dosage is of fundamental importance in animals and plants, exemplified by X chromosome inactivation and dosage compensation. The \"triploid block\" is a classical example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number 1,2. This barrier acts in the endosperm, an ephemeral tissue that nurtures the developing embryo and induces the abortion of hybrid seeds through a yet unknown mechanism. Interploidy hybridizations involving diploid (2x) maternal parents and tetraploid (4x) pollen donors cause failure in endosperm cellularization, leading to embryo arrest 3. Here we show that paternal epigenetically activated small interfering RNAs (easiRNAs) are responsible for the establishment of the triploid block-associated seed abortion in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). We propose that excess of paternally derived easiRNAs in diploid pollen prevents establishment of DNA methylation, leading to triploid seed abortion. Our data further suggest that easiRNAs form a quantitative signal for chromosome number and their balanced dosage is required for post-fertilization genome stability and seed viability.

plant biology↗

bioRxiv2017

microRNA-triggered transposon small RNAs mediate genome dosage response

Chromosome dosage plays a significant role in reproductive isolation and speciation in both plants and animals, but underlying mechanisms are largely obscure1. Transposable elements can promote hybridity through maternal small RNA2, and have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here, we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of LTR-retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21 to 22-nucleotide small RNA in a dose dependent fashion via RNA polymerase IV. We show that these epigenetically activated small-interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (\"the triploid block\"), and that natural variation for miR845 may account for \"endosperm balance\" allowing formation of triploid seeds. Targeting the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

plant biology↗