Biology subjects

Pedor, J. K.

Publications and source records attributed to Pedor, J. K..

3 recordsLinked to original sources

bioRxiv2026

Atlas of stress-induced changes in yeast tRNA modification levels

Transfer RNA (tRNA) modifications are essential for accurate translation and cellular adaptation to environmental changes. Although short-term modification dynamics are well documented, the impact of prolonged stress exposure on the global tRNA landscape remains largely unexplored. Here, we provide the first systematic profiling of tRNA modifications in Saccharomyces cerevisiae following long-term exposure to distinct stress types, including heat, suboptimal pH, oxidative stress (paraquat and diamide), osmotic stress (NaCl and KCl), and genotoxic stress (MMS). Using our broad-range UPLC-MS protocol, we characterized relative nucleoside modification changes across the global tRNA landscape, revealing that long-term stress triggers a global reprogramming of the tRNA epitranscriptome in a stress-specific and time-dependent manner. Remarkably, we identified that pH stress and paraquat induce a near-complete loss of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) modification, and we observe an increase in the non-thiolated 5-methoxycarbonylmethyl (mcm5U) precursor at pH 7. This coupled response is akin to that previously reported for temperature-dependent thiolation deficiency. However, the impact on thiolation is transient in the case of pH stress, but not with paraquat, suggesting two distinct stress-dependent impairment mechanisms of the thiolation pathway. To further integrate our results, we sought to normalize changes in nucleoside modification levels against potential alterations in the tRNA pool. Thus, we performed MarathonRT-based tRNA sequencing and devised the modification deviation (MDm) index. This established that the observed modification changes occurred independently of tRNA isoacceptor abundance, implying that tRNA modification levels are predominantly affected by other factors. Together, this study provides a comprehensive atlas of tRNA modification dynamics under prolonged stress, addressing a critical gap in our understanding of RNA-based translational control. Furthermore, we present the MDm index as a robust quantitative framework to decouple the influence of tRNA abundance from global modification signals, providing a necessary metric for the field to interpret epitranscriptomic reprogramming.

molecular biology↗

bioRxiv2026

Queuosine modification mediates cold-active growth in Shewanella glacialimarina

Efficient protein synthesis in cold-active bacteria requires precise coordination within the translation machinery to overcome the kinetic challenges imposed by growth at near-freezing temperatures (< 5 {degrees}C). Post-transcriptional modifications (PTMs) on transfer RNA (tRNA)--particularly those located at the wobble position 34--are central to this coordination as they regulate decoding speed and fidelity. Here, we show that queuosine (Q) modification in the cold-active marine bacterium Shewanella glacialimarina TZS-4T is dynamically regulated in response to bacterial growth and environmental conditions. Importantly, we demonstrate that Q levels are modulated in a tRNA isoacceptor-specific manner during cold-active growth, while Q-deficiency produces a cold-sensitive phenotype that underscores the functional importance of Q modification. Proteomic analysis of the Q-deficient {Delta}tgt mutant revealed that tRNAHis Q-hypomodification activates the histidine biosynthesis pathway, whereas the concomitant phosphate-starvation-like response reflects a general consequence of global disruption of Q-modified tRNAs. Consequently, we propose a model where Q modification maintains efficient codon decoding and protein quality control at near-freezing temperatures, whereas loss of Q destabilizes codon decoding and ultimately compromises protein homeostasis.

microbiology↗

bioRxiv2026

In-house produced MarathonRT comparison to uMRT and Induro in tRNA sequencing library preparation

Over the past decade, groundbreaking discoveries have cemented transfer RNAs (tRNAs) as versatile regulators of translation and cellular function. As tRNA research gains momentum, several high-throughput sequencing methods have emerged for quantitative analysis of tRNA isoacceptors in cells. However, the strong secondary structure and rich post-transcriptional modification of most tRNA molecules pose significant challenges for reverse transcriptases, thus hampering library preparation and introducing quantification biases. Current approaches rely on processive next generation reverse transcriptases (ngRTs)--they successfully overcome these problems, albeit with the potential caveat of high experimental costs. Here, we introduce a recombinant MarathonRT (MRT) protein both with and without a C-terminal chitin binding domain (CBD), for which we present a simple and robust one-step purification protocol that yields over 26,000 enzymatic reactions per 0.5 L of expression culture. We also developed an affordable colorimetry-based method for determining the specific activity of these enzymes. Importantly, we benchmarked our in-house produced MRT and MRT-CBD enzymes using the mim-tRNAseq workflow and show that their performance match that of commercially available ngRTs. In addition, we implemented the use of a rapid tRNA spin column-based extraction method for tRNA-seq and LC-MS applications, establishing it as a viable alternative to conventional gel-extraction. Combined, this improved workflow significantly reduces the time and cost of tRNA-seq library preparation while providing an easily implementable MarathonRT purification protocol.

molecular biology↗