Biology subjects

Gregorova, P.

Publications and source records attributed to Gregorova, P..

10 recordsLinked to original sources

bioRxiv2026

Ribosome profiling reveals translational dynamics between an archaeal virus and its host

Host-virus interactions remain poorly understood in archaea, whose information processing systems harbor a mosaic of bacterial and eukaryotic features. Here, we used complementary multiomics to investigate interactions between Haloferax tailed virus 1 (HFTV1) and the halophilic archaeon Haloferax gibbonsii (Hg), capturing global translational and transcriptional dynamics across the infection cycle. This revealed co-regulatory expression patterns across HFTV1 and Hg genes. Post-transcriptional regulation was observed for several early HFTV1 genes and the late HFTV1 gene gp17, and the HFTV1 tRNA co-sedimented with Hg ribosome complexes. Infection reshaped the translational landscape of Hg, causing a slow decline in information processing activity, a complex transcriptional response, and amino acid starvation. An unannotated Tat-dependent fimbrial system of Hg was co-regulated with early HFTV1 genes and was correlated with increased biofilm formation, suggesting a potential antiviral defense system. These findings provide high-resolution insight into archaeal host-virus interactions at the evolutionary interface between bacteria and eukaryotes.

microbiology↗

bioRxiv2026

Human models of GLE1 associated LCCS1 reveal neural crest deficiency and multisystem developmental failure accompanied by altered RNA metabolism

Lethal congenital contracture syndrome 1 (LCCS1) is a neurodevelopmental disorder caused by GLE1 c.432-10A>G variant and presenting fetal akinesia, defects in anterior horn spinal cord, skin, skull, and skeletal muscle development. The uniform prenatal lethality of LCCS1 limits access to patient material, thereby hindering mechanistic studies in physiologically relevant models. To overcome this, human embryonic stem cells (hESCs) carrying the LCCS1 variant, patient-derived fetal fibroblasts, and transcriptomic and proteomic profiling were utilized to examine early GLE1 dysfunction in human cells and tissues. Across LCCS1 cell types, reduced global transcription and translation were observed, while nucleocytoplasmic poly(A)+ RNA distribution was unchanged. Despite its context-dependent effects on proliferation, LCCS1 variant altered mRNA decay kinetics and increased stress granule formation in differentiated cells. LCCS1 hESCs retained core pluripotency but reduced choline acetyltransferase and {beta} tubulin III levels, together with increased neurofilament inclusion incidence, indicate functional immaturity in differentiated spinal motor neurons. Differentiation of LCCS1 hESC-derived gastruloids uncovered broad perturbations in neuromuscular and neural crest derivative development, results which were supported by phenotypes detected in ectodermal organoids and fetal LCCS1 tissue. These findings provide new mechanistic insight into LCCS1 pathogenesis and establish a robust human model framework for studying neurodevelopmental disorders.

developmental biology↗

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↗

bioRxiv2026

Queuosine promotes wecB-dependent phage resistance and biofilm formation in marine bacterium Shewanella glacialimarina

Transfer RNA (tRNA) modifications critically fine-tune translational accuracy and efficiency, influencing bacterial adaptation to environmental challenges. Among these, the queuosine (Q) modification has recently emerged as a regulator of biofilm formation, yet its role during phage infection remains unknown. Here, we investigate how Q modification links host translational control to phage infection. We show that phage infection activates the Q biosynthetic pathway, leading to elevated Q levels and enhanced translation of NAT-biased genes. This shift drives two interconnected outcomes, namely increased biofilm formation and enhanced mutagenesis mediated by translesion synthesis polymerases. We further identify conserved, slippage-prone regions within surface-related genes that act as hotspots for adaptive variation. Together, our findings uncover a novel mechanistic link between tRNA modification and phage-driven bacterial diversification. AUTHOR SUMMARYTo survive changing environmental conditions, bacteria constantly adjust their metabolic processes, including how they translate their genetic information into proteins. One such adjustment mechanism involves carefully balancing the level of chemical modification on transfer RNA (tRNA), the key adapter molecule that carries amino acids but also regulates protein synthesis. In this study, we explore how a cold-active bacterium relies on queuosine, a wobble position tRNA modification, to mediate host cell responses to bacteriophage infection. We observe that as viral infection progresses, the bacterial host cells increase the level of queuosine modification present on tRNAs. This alters the efficiency by which specific proteins are produced, favoring those that are involved in biofilm synthesis and thereby form protective communities that help bacteria survive stress. At the same time, queuosine also promotes error-prone DNA replication processes that lead to an overall increase in bacterial mutation rates. Taken together, our results reveal how a small molecular change in tRNA can reshape bacterial responses to viral infection, ultimately driving genetic diversity and survival.

microbiology↗

bioRxiv2025

The halophilic archaeon Halogranum roseipondis sp. nov. is susceptible to a virus carrying an exceptionally high number of viral tRNA genes

Archaea constitute a diverse group of organisms, many of which inhabit extreme environments, such as haloarchaea that dominate hypersaline ecosystems, like solar salterns. Although sampling of solar salterns and other hypersaline environments have resulted in numerous haloarchaeal isolates, including 3 classified and 25 unclassified Halogranum species, no complete genome has been reported for any member of this genus. Here we present the first comprehensive study of Halogranum sp. SS5-1 isolated from a solar saltern in Samut Sakhon, Thailand. Hgn. SS5-1 is a pleomorphic, aerobic heterotroph that thrives in high salinity and moderate temperature and is capable of hydrolysing starch. Its genome consists of a 3.6 Mbp chromosome and seven additional plasmids. Based on our phylogenetic analyses, which establish Hgn. SS5-1 as a distinct species, we propose that it will be classified as the novel species Halogranum roseipondis sp. nov. SS5-1T. Additionally, we report that Hgn. roseipondis sp.nov. SS5-1T is infected by Hagravirus capitaneum (HGTV-1), the only virus known to infect a Halogranum host. HGTV-1 exhibits a unique head-tailed morphology and encodes the largest archaeal virus dsDNA genome known to date, including 34 tRNA encoding genes. Codon usage analysis of the viral genome suggests partial alignment with host preferences, yet the abundance of viral tRNA genes hints to broader roles, potentially including translation modulation. This study establishes Hgn. roseipondis and HGTV-1 as a novel virus-host system, opening avenues to explore infection dynamics and the roles of virus-encoded tRNA in archaea. ImportanceArchaea that thrive in high-salinity environments are key players in geochemical cycles and the primary producers in their habitat. Despite their ecological significance and importance for the development of novel applications in synthetic biology, haloarchaea remain poorly studied. For example, exploration of haloarchaea is required to understand the evolution of cellular complexity and the molecular mechanisms that allow cells to thrive in harsh environmental conditions. In this study, we characterize a novel archaeon, Halogranum roseipondis sp. SS5-1T, alongside the infection cycle of its associated virus, Hagravirus capitaneum. This tailed myovirus carries an extraordinary set of 34 viral tRNA genes, a feature that opens intriguing questions about virus-host interactions and translational control. Our findings lay the groundwork for future investigations into the expression and function of viral tRNAs in an archaeal model system, thereby enabling the study of archaeal translation and virus-driven modulation of host cellular processes.

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↗

bioRxiv2024

Purification of micrococcal nuclease (MNase) for use in ribosomal profiling of high-salinity extremophiles

Nucleases, i.e. enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids, are essential tools in molecular biology and biotechnology. Staphylococcus aureus nuclease (MNase) is particularly interesting due to its thermostability and Ca2+-dependence, making it the prime choice for applications where nuclease modulation is critical, such as ribosome profiling in bacteria and halophilic archaea. The latter poses a technical and economical challenge: high salt reaction conditions are essential for maintaining ribosome integrity but negatively impact the MNase activity, necessitating using large amounts of nuclease to achieve efficient cleavage. Here, we set out to generate an optimized production protocol for two forms of MNase -- fully processed MNaseA and the 19 amino acid propeptide-containing MNaseB -- and to biochemically benchmark them against a commercial nuclease. Our results show that both MNases are highly active in normal reaction conditions, but MNaseA maintains higher enzymatic activity in high salt concentrations than MNaseB. MNaseA also retains >90% of its activity after multiple freeze-thaw cycles when stored at -80 {degrees}C in a buffer containing 5% glycerol. Importantly, ribosome profiling experiments in the haloarchaeon Haloferax volcanii demonstrated that MNaseA produces ribosome footprints and hallmarks of active translation highly comparable to those obtained with the commercial nuclease, making it a suitable alternative for high-salt ribosome profiling applications. In conclusion, our method can be easily implemented for efficient MNaseA production, thereby providing access to an effective, robust, and cost-efficient alternative to commercial nucleases, as well as facilitating future translation studies into halophilic organisms.

biochemistry↗

bioRxiv2024

Deciphering the RNA Modification Landscape in Arabidopsis Chloroplast tRNAs and rRNAs Reveals a Blend of Ancestral and Acquired Characteristics

Chloroplasts in plant leaves are essential for protein synthesis, relying on transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) encoded by the chloroplast genome. Although post-transcriptional modifications of these non-coding RNAs are common in many systems, chloroplast tRNA and rRNA modifications are not well characterised. In this study, we investigated the post-transcriptional modifications in chloroplast tRNAs and rRNAs of Arabidopsis thaliana using tRNA sequencing, liquid chromatography-mass spectrometry, targeted rRNA sequencing, and analysis of public data. Our results revealed similarities between chloroplast non-coding RNAs and bacterial systems (e.g., Escherichia coli), such as modification patterns at the anticodon-adjacent position and the variable loop of tRNAs, along with conserved modifications in the small subunit rRNA. Additionally, we identified features shared with eukaryotic systems that likely contribute to the correct three-dimensional structure of chloroplast tRNAs. Unique modifications were also discovered, including a potential novel modification at wobble position in tRNA-IleCAU, which may be crucial for distinguishing isoleucine codons from methionine codons, and chloroplast-specific rRNA modifications that likely compensate for altered ribosome structure. These findings suggest that the chloroplast translation machinery, through co-evolution with its eukaryotic host, has adopted features beyond those typically found in bacteria, reflecting a blend of ancestral and acquired characteristics.

plant biology↗

bioRxiv2023

Global analysis of aging-related protein structural changes uncovers enzyme polymerization-based control of longevity

Aging is associated with progressive phenotypic changes over time. Virtually all cellular phenotypes are produced by proteins and structural alterations in proteins can lead to age-related diseases. Nonetheless, comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contribution to age-related phenotypes is lacking. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry. The results, compiled in online ProtAge-catalog, unravelled age-related functional changes in regulators of translation, protein folding and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction and led to life span extension. Altogether, this comprehensive map of protein structural changes enables identifying novel mechanisms of age-related phenotypes and offers opportunities for their reversal.

cell biology↗

bioRxiv2020

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Post-transcriptional RNA modifications play an important role in cellular metabolism with homeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukemia and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9 min sample-to-sample run. To validate the performance and versatility of our method, we analyzed tRNA modification patterns of representative microorganisms from each kingdom of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae) and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homeostasis studies of RNA modification in complex biological samples.

molecular biology↗