Biology subjects

Wesseling, H.

Publications and source records attributed to Wesseling, H..

4 recordsLinked to original sources

bioRxiv2025

Purity and stability of modified nucleosides in the context of accurate quantification by LC-MS

Accurate LC-MS (liquid chromatography coupled mass spectrometry) analysis of RNA modifications relies on synthetic nucleoside standards whose chemical integrity determines both qualitative identification and quantitative measurements. While the purity of these standards is typically verified prior to use, their long-term chemical stability during storage has not been systematically investigated. Here, we evaluated the stability of 44 canonical and modified ribonucleosides in aqueous solution during storage at -80 {degrees}C and -20 {degrees}C. Initial quality control confirmed the identity of all tested standards and revealed purity issues in selected compounds, including contamination of 1-methyladenosine (m1A) with 6-methyladenosine (m6A) and the presence of S- and R-isomers of 5-(carboxyhydroxymethyl)-2'-O-methyluridine (mchm5Um). Long-term LC-UV-MS monitoring over 12 months showed that 30 nucleosides remained stable, two were stable for at least six months, and 12 exhibited substantial quantitative changes. Seven nucleosides formed detectable degradation products, most of which could be structurally assigned. Quantum-chemical calculations of reaction free energies for deglycosylation, deamination, deacetylation and desulfurization correlated with the experimentally observed stability trends. Based on these results, we propose a practical guideline for the preparation, storage and analytical quality control of nucleoside standards, including recommendations for purity verification by UV spectroscopy and quantitative NMR. These guidelines provide an experimental framework to improve the robustness and inter-laboratory comparability of LC-MS-based RNA modification analysis.

molecular biology↗

bioRxiv2025

Temporal and spatial profiling of ALKBH5 activity through NAIL-MS and compartmentalized RNA Isolation

RNA modifications, especially m6A in human mRNA, are believed to be dynamically regulated through RNA writers and erasers. The key eraser of m6A is ALKBH5 with its function well proven in vitro, while in vivo evidence is lacking. Here, we set out to exploit nucleic acid isotope labelling coupled mass spectrometry (NAIL-MS) in a pulse chase set-up to study the in vivo function of ALKBH5 on human RNAs. For this we purified poly(A) from whole cell total RNA and found that, steady-state m6A levels and turnover dynamics were nearly identical between WT and ALKBH5 KO, despite clear evidence of robust RNA turnover within an 8-hour labeling period. To assess whether ALKBH5 might act in a compartment-specific manner, we employed an advanced subcellular fractionation strategy, allowing for the isolation of chromatin-associated, nucleoplasmic, and cytoplasmic RNA. These analyses confirmed that m6A accumulates during transcript maturation, with levels peaking in nuclear fractions and decreasing following export to the cytoplasm, supporting the thesis m6A is a dynamic modification. Notably, however, spatial and temporal profiles of m6A distribution and decay were unaffected by ALKBH5 KO. Even in chromatin-associated and nucleolar mRNA, where co-transcriptional modification and potential demethylation would be most plausible, m6A dynamics remained indistinguishable between WT and KO cells in the NAIL-MS context. We thus conclude that ALKBH5 has no major role in mRNA m6A demethylation in HEK 293T cells grown under optimal conditions.

molecular biology↗

bioRxiv2021

Phosphorylation-dependent control of Arc protein by synaptic plasticity regulator TNIK

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate-early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arcs subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders.

neuroscience↗

bioRxiv2018

Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

Translation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties.

neuroscience↗