Biology subjects

Sarin, L. P.

Publications and source records attributed to Sarin, L. P..

4 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↗

bioRxiv2025

Rapid tRNA Isolation and Chemiluminescent Northern Blot Detection of tRNA and tRNA-Derived Fragments

Transfer RNA (tRNA), its post-transcriptional modifications, and tRNA-derived fragments (tRFs) play essential roles in cellular processes and gene regulation. Here, we present a fast and efficient tRNA isolation using silica spin columns. To analyze the isolated tRNA and detect tRFs, we describe a sensitive and cost-effective non-radioactive Northern blotting technique. Additionally, this blotting method is compatible with chemical affinity modifiers, such as [p-(N-acrylamino)-phenyl]mercuric chloride (APM) or 3-(acrylamido)phenylboronic acid (APB) enabling the detection of chemical modifications in specific tRNA isoacceptors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/681074v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1b0618forg.highwire.dtl.DTLVardef@d507a4org.highwire.dtl.DTLVardef@1e8b6faorg.highwire.dtl.DTLVardef@1450326_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIRapid (<30 min) tRNA isolation from total RNA using silica containing spin columns C_LIO_LIProcedure for DNA probes biotinylation via terminal deoxynucleotidyl transferase C_LIO_LINon-radioactive chemiluminescent Northern blotting C_LI

molecular biology↗

bioRxiv2025

An efficient one-step rRNA depletion method for RNA sequencing in non-model organisms

RNA sequencing (RNA-seq) has revolutionized global transcriptomic analysis, ribosome footprinting, and polysome profiling, providing a wealth of data. Importantly, many RNA-based omics approaches typically involve either the removal of ribosomal RNA (rRNA) or selection of messenger RNA (mRNA) prior to sequencing, thereby enriching reads that map to the translationally active part of the transcriptome. Prokaryotic mRNA differs from eukaryotic mRNA in that it lacks the 3 polyadenylated tail, which excludes the use of poly(A)-based selection methods. While commercial rRNA depletion products exist for a growing number of prokaryotes, their proprietary nature and potential inefficiency with non-model organisms are factors that may limit broad-scale application. To mitigate this issue, we designed DepStep, a consolidated workflow for one-step rRNA depletion using species-specific biotinylated antisense probes for selective hybridization and removal of the target rRNA molecules. As a proof-of-concept, RNA-seq libraries of the psychrophilic gram-negative bacterium Shewanella glacialimarina TZS-4T were prepared using both DepStep and a commercial rRNA depletion kit for gram-negative bacteria, to which DepStep was benchmarked. DepStep compares favorably to the commercial depletion kit; it efficiently removes >98.6% of the rRNA content, and a slight increase in total read counts aligning to the coding sequences (CDS) was observed. Importantly, DepSteps cost-per-sample is three times lower than the commercial kit, establishing DepStep as a simple yet cost-effective alternative to commercial solutions.

molecular biology↗