Biology subjects

Koutmou, K. S.

Publications and source records attributed to Koutmou, K. S..

9 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↗

bioRxiv2026

Mapping the rRNA methylome reveals contributions of methyltransferases to ribosome function and antibiotic sensitivity

Ribosomal RNA (rRNA) folds into a complex structure used as the macromolecular core for protein synthesis. Chemical modification of rRNA contributes to ribosome structure, function, and susceptibility to antibiotics. Despite their importance, the enzymes responsible for specific rRNA modifications remain unknown in most species. In this work, we integrate genetics and biochemistry with sequencing and mass spectrometry to uncover enzymes responsible for rRNA methylation events in Bacillus subtilis. We characterize 17 enzymes responsible for 20 methylation modifications on the 16S and 23S rRNAs, 11 of which are encoded by previously uncharacterized genes. For each rRNA methyltransferase, we define the modification identity, location, and we determine the impact of loss of cognate rRNA methylation on ribosome biogenesis and antibiotic sensitivity. Our findings demonstrate that loss of nearly half of the 17 genes studied results in alterations to ribosome assembly or antibiotic sensitivity underscoring the importance of chemical modifications to ribosome function.

biochemistry↗

bioRxiv2026

RNase 4 improves bottom-up modification mapping of E. coli total tRNAs using HILIC-MS/MS

The structural and functional diversity of RNAs is expanded by the post-transcriptional incorporation of nucleoside variants. Emblematic of this, tRNAs contain extensive modifications that ensure their function during protein synthesis. Mass spectrometry has long been the field standard for identifying specific sites of chemical modifications on RNA. Nonetheless, mass spectrometry-based mapping approaches are not widely implemented. This is partially due to technical challenges associated with current methodologies including the limited diversity of available RNases, complexity of RNA mixtures, and conventional use ion-pairing reagents that require dedicated instrumentation. Here, we present a bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow employing hydrophilic interaction liquid chromatography (HILIC) without ion-pairing reagents to globally map E. coli tRNA modifications. We implement orthogonal digestions using RNase 4 and a folded digestion scheme with RNase T1 to generate uniquely mappable oligonucleotides compatible with HILIC-MS/MS analysis and achieve 75-100% sequence coverage for most tRNA isoacceptors. HILIC-MS/MS matches the performance of traditional ion-pairing reverse-phased LC-MS/MS. This level of coverage allowed us to discover a new site of methylation (Gm17) in tRNAGly, and confirm the presence of an s4U8 modification predicted in tRNAArg. Furthermore, by applying this method to E. coli lacking the m5U54 methyltransferase (trmA) we confirmed the established dependence of acp3U47 insertion on m5U54 in tRNAPhe. Our findings show that RNase 4 improves bottom-up tRNA sequencing, enabling high-quality E. coli tRNA analysis without ion-pairing reagents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/727428v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1f483a3org.highwire.dtl.DTLVardef@1ee5e01org.highwire.dtl.DTLVardef@5db88borg.highwire.dtl.DTLVardef@fee855_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Pseudouridylation landscape across 42 S. cerevisiae cytosolic tRNA isoacceptors via Nanopore direct RNA sequencing

Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet, in the widely used model organism, the budding yeast Saccharomyces cerevisiae, tRNA pseudouridine sites have not been comprehensively annotated. Here, we performed two high-throughput methods established for detecting tRNA pseudouridylation positions: Nanopore direct RNA sequencing (DRS) and 2-bromoacrylamide-assisted cyclization sequencing (BACS). Using DRS, we sequenced cytosolic tRNA from seven pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-two existing pseudouridine annotations and predicted an additional ten previously unannotated pseudouridine sites. Our analysis also revealed DRS signal changes at several non-uridine sites with the loss of a PUS, including apparent changes in modification abundances at position 37 upon deletion of Pus3. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3, underscoring the need for caution in interpreting DRS-based signal changes. Using BACS, we confirmed the ten DRS-predicted novel sites, while further detecting eleven additional previously unannotated pseudouridine sites. Combining existing modification annotations from the Modomics database with our DRS and BACS datasets, we created a map of all detected pseudouridines --totaling 126 sites, including 21 novel sites--and the enzymes responsible for their catalysis, across the forty-two nuclear-encoded S. cerevisiae tRNA isoacceptors.

molecular biology↗

bioRxiv2025

Cytoplasmic localization of PUS7 facilitates a pseudouridine-dependent enhancement of cellular stress tolerance

Pseudouridine ({Psi}) is an abundant post-transcriptional modification found across all classes of RNA. It has been widely speculated that {Psi} inclusion in mRNAs might provide an avenue for cells to control gene expression post-transcriptionally. Here we demonstrate that one of the principal mRNA pseudouridylating enzymes, pseudouridine synthase 7 (PUS7), exhibits a stress-induced accumulation in the cytoplasm of yeast and human epithelial lung cells. Stress-induced and cytoplasmic localization of PUS7 promote {Psi}-incorporation into hundreds of mRNA targets. Furthermore, engineered PUS7 cytoplasmic localization increases cellular fitness under ROS and divalent metal ion stress. Consistent with this, transcripts modified upon PUS7 cytoplasmic localization are enriched within mRNAs encoding proteins involved in divalent metal metabolism and ROS stress pathways. In contrast, tRNA sites modified by PUS7 ({Psi}13 and {Psi}35 are unperturbed). Quantitative proteomics reveal a reshaping of the proteome upon PUS7 relocalization under stress, with proteins involved in metal and ROS homeostasis being particularly sensitive to PUS7 localization. Collectively, our data demonstrate that PUS7 localization alters mRNA pseudouridylation patterns to modulate protein production and enhance cellular fitness.

biochemistry↗

bioRxiv2025

TlyA is a 23S and 16S 2'-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis

Ribosomal RNA (rRNA) is methylated in organisms ranging from bacteria to metazoans. Despite the pervasiveness of rRNA methylation in biology, the function of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2'-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside antibiotics that target the 16S rRNA. We show that{Delta} tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach and based on sequence alignments with other rRNA methyltransferases we tested the importance of potential catalytic residues and S-adenosyl-L-methionine (SAM) cofactor binding sites. We show that TlyA shares the common rRNA methyltransferase catalytic triad KDK and a SAM binding motif GxSxG which differs from Mtb TlyA. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since E. coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb and more divergent Gram-negative bacteria providing new insight into translation and antibiotic resistance mechanisms.

microbiology↗

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↗

bioRxiv2022

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid and accurate protein production. The inclusion of chemically modified bases into mRNAs has the potential to alter the strength and pattern of hydrogen bonding between mRNAs and aminoacyl-tRNAs to alter protein synthesis. We investigated how the Nl-methylpseudouridine (m1{Psi}) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the ability of codons to react with cognate and near-cognate tRNAs and release factors. We find that the presence of a single m1{Psi} does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1{Psi} can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1{Psi}, and the related naturally occurring pseudouridine ({Psi}) modification, exhibit the ability to both increase and decrease the extent of amino acid misincorporation in a codon-position and tRNA dependent manner. To ascertain the chemical logic for our biochemical and cellular observations, we computationally modeled tRNAIle(GAU) bound to unmodified and m1{Psi}- or {Psi}-modified phenylalanine codons (UUU). Our modeling suggests that changes in the energetics of mRNA:tRNA interactions largely correlate with the context specificity of Ile-miscoding events we observe on {Psi} and m1{Psi} containing Phe codons. These studies reveal that the sequence context of a given modification within an mRNA plays a large role in determining how (and if) the modification impacts the number and distribution of proteoforms synthesized by the ribosome.

biochemistry↗

bioRxiv2022

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

biochemistry↗