Biology subjects

Tardu, M.

Publications and source records attributed to Tardu, M..

3 recordsLinked to original sources

bioRxiv2026

Integrating mass spectrometry with Nanopore direct RNA sequencing for de novo modification profiling of bacteriophage MS2

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both LC-MS/MS and Nanopore DRS. LC-MS/MS analysis revealed that {Psi} is the predominant modification, present at approximately one site per RNA molecule, while all other modifications were found at low levels (< 0.04 modifications per RNA). In contrast, Dorado-based modification calling not only confirmed {Psi} but also predicted multiple m6A, m5C, 2OMe, and inosine sites not supported by LC-MS/MS data. To refine modification calling, we used in vitro transcribed (IVT) RNA as a negative control and subtracted IVT-derived false positive rates from native RNA predictions. This adjustment reduced overcalling and improved confidence in site-specific predictions. These data demonstrate that while Dorado can detect RNA modifications de novo, its predictions require careful filtering and validation. Our studies of provide a rare benchmark dataset for assessing the ability of transcriptome-wide methods to accurately identify RNA modifications and estimate modification stoichiometry. These findings support the use of orthogonal approaches, including LC-MS/MS and IVT controls, alongside Nanopore sequencing to provide a more reliable and interpretable strategy for studying RNA modification patterns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/729303v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@637a55org.highwire.dtl.DTLVardef@f48272org.highwire.dtl.DTLVardef@a0465aorg.highwire.dtl.DTLVardef@6af045_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in E. coli, Trm2 in S. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2{Delta} cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

biochemistry↗

bioRxiv2018

N4-acetylcytidine and 5-formylcytidine are present in Saccharomyces cerevisiae mRNAs

Post-transcriptional nucleoside modifications have long been recognized as key modulators of non-coding RNA structure and function. There is an emerging appreciation that the chemical modification of protein-coding messenger RNAs (mRNAs) also plays critical roles in the cell. Although there are over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. We sought to identify and quantify modifications present in the mRNAs of yeast cells using a high throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that measures the levels of 36 types of RNA nucleosides in parallel. We detected the presence of six modified nucleosides in mRNAs at relatively high abundances: N7-methylguanosine, N6-methyladenosine, 2-O-methylguanosine, 2-O-methylcytosine, N4-acetylcytidine and 5-formylcytidine. Additionally, we investigated how the levels of mRNA modifications vary in response to cellular stress. We find that the concentrations of mRNA modifications including N6-methyladenosine and N4-acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and utilizes a high-throughput approach to search for modifications that highlights the value of integrating mass-spectrometry tools in the mRNA modification discovery and characterization pipeline.

biochemistry↗