bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.05.09.653160

Concurrent detection of chemically modified bases in yeast mitochondrial tRNAs by Nanopore direct RNA sequencing

Reinsch, J. L.·Garcia, D. M.
View original source ↗Export metadata ↓

Abstract

Saccharomyces cerevisiae is an invaluable model in the study of mitochondrial tRNA biology. Yet the positions of modified bases in all yeast mitochondrially-encoded tRNAs (mt-tRNAs) are still not fully mapped. We performed Nanopore direct RNA sequencing (DRS) on tRNAs from the crude mitochondrial fraction of yeast to map base modifications across all 24 mt-tRNA isoacceptors. Additionally, we developed a method to detect dihydrouridine sites in tRNAs, tD-seq, where chemical reduction of dihydrouridine causes disruptions to reverse transcription. We mapped dihydrouridine, pseudouridine, and N2-dimethylguanosine sites in mt-tRNAs using DRS, tD-seq, and knockouts of five conserved tRNA-modifying enzymes. Our results establish Dus1 and Dus2 as the enzymes responsible for D14, D16, D17, D17a, and D20 formation in S. cerevisiae mt-tRNAs, and revealed interactions between Dus1, Dus2, and Trm1-catalyzed modifications. We provide a comprehensive analysis of S. cerevisiae mt-tRNA base modifications, and identify novel modification "circuits" in yeast mt-tRNAs, in which the loss of a single enzymes activity can change modification levels at sites catalyzed by other enzymes. These findings expand our understanding of mt-tRNA base modifications and their interdependence, and advance opportunities for the yeast model for investigating defects in human mt-tRNA function. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/653160v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@9b7315org.highwire.dtl.DTLVardef@1a089dcorg.highwire.dtl.DTLVardef@bb01b7org.highwire.dtl.DTLVardef@16dd34d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Reinsch, J. L., Garcia, D. M.. 2025-05-10. Concurrent detection of chemically modified bases in yeast mitochondrial tRNAs by Nanopore direct RNA sequencing. https://doi.org/10.1101/2025.05.09.653160

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Integrated Spatial Metabolomics and Proteomics from the Same Tissue Section Using a Conductive ITO-PET Slide

Integrating spatial metabolomics and spatial proteomics on the same tissue section remains challenging because matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-based proteomics impose different requirements on sample slides. Here, we developed and systematically evaluated a conductive indium tin oxide-coated polyethylene terephthalate (ITO-PET) slide that enables sequential MALDI-MSI and LCM-liquid chromatography-mass spectrometry (LCM-LC-MS) analysis of the same tissue section. Using mouse brain tissue as a model, ITO-PET provided MALDI-MSI performance closely comparable to conventional ITO-glass, including spectral concordance (Pearson correlation, R = 0.90), ion detection coverage, metabolite annotation, signal intensity distribution, and preservation of spatial molecular patterns. Following MALDI-MSI, the ITO-PET slide enabled cutting-mode LCM and yielded proteomic signal intensities and numbers of identified protein groups comparable to those obtained with conventional PEN-glass slides. Across different tissue sampling areas, proteomic signal intensity distributions, precursor ion counts, and protein group identifications remained broadly comparable before and after MALDI-MSI, with substantial overlap in identified protein groups. Similar patterns were observed in mouse kidney, lung, spleen, and liver tissues, further supporting the applicability of the workflow across different tissue types. By combining the electrical conductivity required for MALDI-MSI with the mechanical properties required for LCM cutting, the ITO-PET slide addresses a major material incompatibility between the two analytical modalities and enables sequential spatial metabolomic and proteomic analysis from the same tissue section. This workflow provides a practical analytical platform for obtaining complementary molecular information from spatially limited biological specimens.

molecular biology↗
bioRxiv2026

Aβ42-Driven α-synuclein Fibril Polymorphism and Distinct Intracellular Aggregation

The frequent coexistence of -synuclein (-syn) and amyloid-{beta} (A{beta}) aggregates in neurodegenerative diseases suggests that heterotypic interactions between these amyloidogenic proteins may influence disease progression, yet their molecular consequences remain poorly understood. Here, we investigated how distinct aggregation states of A{beta}42, monomers and preformed fibrils (PFFs), modulate -syn fibril formation, structure, and downstream neuronal pathology. Thioflavin T kinetics showed that A{beta}42 monomers delayed -syn fibril formation, whereas A{beta}42 PFFs exhibited a trend toward accelerated aggregation, indicating aggregation state-dependent effects on -syn aggregation. Negative-stain TEM, proteinase K digestion, and solid-state NMR spectroscopy further demonstrated that both A{beta}42 monomers and PFFs altered -syn fibril structure, generating distinct fibril conformations depending on the A{beta}42 concentration and aggregation state. To determine whether these conformational differences influence pathological activity, -syn PFF variants generated in the presence of different concentrations of A{beta}42 monomers or PFFs were applied to dopaminergic neuronal cells. -Syn fibrils formed in the presence of A{beta}42 PFFs showed greater capacity to induce intraneuronal -syn aggregation than -syn PFFs, whereas fibrils formed in the presence of A{beta}42 monomers exhibited similar or reduced seeding capacity relative to -syn PFFs. Together, our findings demonstrate that heterotypic interactions with A{beta}42 reshape -syn aggregation pathways and fibril conformations, generating structurally distinct -syn fibril populations with different neuronal seeding activities. These results provide a molecular framework for understanding how cross-talk between amyloidogenic proteins may contribute to structural and pathological heterogeneity in mixed neurodegenerative diseases.

molecular biology↗
bioRxiv2026

Insulators As Dynamic, Tunable Regulators of Enhancer-Promoter Coordination in Living Drosophila Embryos

The three-dimensional organization of the genome enables enhancers and promoters to interact across vast distances and direct transcription. Yet whether architectural elements, such as insulators, serve as rigid, passive barriers or as dynamic, active organizers of this communication remains unclear. Here, using single-cell, live imaging of a Drosophila transgene in which a single enhancer regulates two equidistant promoters, we confirm that the enhancer engages both promoters simultaneously and show that coordinated bursting is intrinsically more productive than uncoordinated activity. Flanking this system with insulators increases coordinated bursting frequency and transcriptional output, indicating that insulator-mediated looping promotes multi-way enhancer-promoter interaction. Further, bidirectionally-paired, homotypic insulators produce stronger coordination than unidirectional pairs. Inserting an intermediate insulator to generate competing loop configurations, together with two-state promoter modeling, we show that these chromatin loops are highly dynamic. This work reframes insulators as active, tunable regulators that shape the frequency, coordination, and productivity of enhancer-promoter communication.

molecular biology↗