Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.02.06.478964

Development of a Ribosome Profiling Protocol to Study Translation in the yeast Kluyveromyces marxianus

Fenton, D. A.·Kiniry, S. J.·Yordanova, M. M.·Baranov, P. V.·Morrissey, J. P.
View original source ↗Export metadata ↓

Abstract

Kluyveromyces marxianus is an interesting and important yeast because of particular traits like thermotolerance and rapid growth, and applications in food and industrial biotechnology. Knowing how K. marxianus responds and adapts to changing environments is important to achieve a full understanding of the its biology and to develop bioprocesses. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. Whereas transcriptome analysis by RNA-Seq quantifies mRNA abundance, ribosome profiling allows codon-resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs and is emerging as a valuable tool to study translation control of gene expression. We report here the development of a ribosome profiling method for K. marxianus and we make the procedure available as a step by step protocol. To aid in the analysis and sharing of ribosome profiling data, we also added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. As many studies only focus on the use of RNA-Seq to study K. marxianus in different environments, the availability of ribosome profiling is a powerful addition to the K. marxianus toolbox. Graphical abstractDevelopment of a Ribosome Profiling protocol to study gene expression in the thermotolerant yeast Kluyveromyces marxianus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/478964v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1268da7org.highwire.dtl.DTLVardef@1ddff1org.highwire.dtl.DTLVardef@1597da4org.highwire.dtl.DTLVardef@47c2ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Fenton, D. A., Kiniry, S. J., Yordanova, M. M., Baranov, P. V., Morrissey, J. P.. 2022-02-07. Development of a Ribosome Profiling Protocol to Study Translation in the yeast Kluyveromyces marxianus. https://doi.org/10.1101/2022.02.06.478964

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

KEEP EXPLORING

Related preprints

bioRxiv2026

LOca-1: A minimal calcium-activated arginyl-specific orthocaspase from the Loki's Castle hydrothermal vent field

Orthocaspases are prokaryotic homologs of metacaspases within the C14 family of cysteine proteases, yet their biochemical properties and cellular roles remain poorly understood. Here, we report the discovery and comprehensive characterization of LOca-1, a metagenome-derived orthocaspase from the Loki's Castle hydrothermal vent field. LOca-1 consists primarily of a single, conserved p20 catalytic domain and undergoes calcium-dependent activation through cleavage at a conserved arginine residue. Once activated, LOca-1 displays strict arginyl-specific proteolytic activity and strong selectivity for residues surrounding the cleavage site. The enzyme is both thermoactive and thermostable, exhibiting maximal activity at 65 {degrees}C and a melting temperature of approximately 72 {degrees}C. Structural modelling revealed conservation of the catalytic core relative to plant metacaspases despite substantial sequence divergence. To our knowledge, LOca-1 is only the second biochemically characterized orthocaspase and the first reported from a deep-sea hydrothermal vent environment. LOca-1 expands the known biochemical diversity of prokaryotic metacaspase homologs and provides insights into the properties of a minimal p20-domain orthocaspase. Together, our findings establish LOca-1 as a thermostable, calcium-activated arginyl-specific orthocaspase and provide a foundation for future studies of orthocaspase function in prokaryotes.

molecular biology↗
bioRxiv2026

A TMEM175 Mutation Disrupts the Lysosomal-Mitochondrial Axis Causing Short QT Syndrome and Sudden Cardiac Death

Short QT Syndrome (SQTS) is a rare cardiac channelopathy characterized by abnormally short QTc intervals and increased sudden cardiac death (SCD) risk. Only 20-30% of SQTS cases have been linked to genetic mutations, typi-cally in surface membrane ion channels. We identified a novel lysosomal TMEM175 gene mutation (p.Arg377His) in 7 patients in a family with SQTS and SCD. Genetic analyses, including whole exome sequencing in the family and rare-variant burden testing in [~]118,000 UK Biobank participants, sug-gested an association between TMEM175 rare variants and QT interval short-ening. Functional studies in CRISPR-engineered mice and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) revealed that TMEM175R377H causes shortened QT intervals and hastened repolariza-tion, respectively, as well an increased arrhythmia susceptibility. Isolated TMEM175R377H cardiomyocytes showed action potential duration (APD) abbre-viation, confirmed in whole TMEM175R377H mouse hearts and patient-specific iPSC-CM monolayers. In silico and electron microscopy studies showed that TMEM175R377H disrupts lysosomal-mitochondrial interactions, leading to mito-chondrial dysfunction, as evidenced by the reduced maximal respiration and spare respiratory capacity, with reduced ATP production, IKATP activation, and APD shortening. This mutation represents the first non-sarcolemmal ion chan-nel defect associated with SQTS and high SCD risk, potentially expanding the genetic landscape of the disease.

molecular biology↗
bioRxiv2026

Protecting host mitochondria with vitamin B3 preserves bioenergetics and thermogenesis during antibiotic treatment

Antibiotics that target bacterial translation can also impair mitochondrial function because mitochondria retain bacterial-like ribosomes. As antibiotic resistance continues to increase and the use of several broad-spectrum antibiotics has been limited by their mitochondrial toxicity, there is a growing need to understand their off-target effects in host physiology, and to identify strategies that can mitigate antibiotic-induced mitochondrial dysfunction. We demonstrate that antibiotic chloramphenicol (CA) induces a dose-dependent reduction in mitochondrial respiration and mitochondrial thermogenesis in Drosophila S2 cells, human HEK293T cells and in developing Drosophila larvae. In vivo, CA exposure triggers female-biased aberrant innate immune activation of hosts carrying naturally occurring mitochondrial rRNA variants. Metabolomic profiling revealed alterations in vitamin B3 metabolism in Drosophila during and following CA exposure. Among the vitamin B3 vitamers examined, nicotinamide (NAM) exhibited the strongest protective effect, mitigating the CA-induced impairments in mitochondrial respiration and thermogenesis, as well as aberrant immune activation. We identify mitochondrial thermogenesis as a previously unrecognized target of antibiotic-induced mitochondrial perturbation and a sensitive marker of mitochondrial stress. Furthermore, we demonstrate that co-supplementation of CA with NAM mitigates antibiotic-induced mitochondrial toxicity through evolutionarily conserved mitochondrial pathways. Finally, we establish Drosophila as a powerful model for investigating antibiotic-induced mitochondrial dysfunction and for developing therapeutic strategies to protect host mitochondria during antibiotic treatment.

molecular biology↗