Biology subjects

Nemudryi, A.

Publications and source records attributed to Nemudryi, A..

7 recordsLinked to original sources

bioRxiv2026

Integrating mass spectrometry with Nanopore direct RNA sequencing for de novo modification profiling of bacteriophage MS2

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both LC-MS/MS and Nanopore DRS. LC-MS/MS analysis revealed that {Psi} is the predominant modification, present at approximately one site per RNA molecule, while all other modifications were found at low levels (< 0.04 modifications per RNA). In contrast, Dorado-based modification calling not only confirmed {Psi} but also predicted multiple m6A, m5C, 2OMe, and inosine sites not supported by LC-MS/MS data. To refine modification calling, we used in vitro transcribed (IVT) RNA as a negative control and subtracted IVT-derived false positive rates from native RNA predictions. This adjustment reduced overcalling and improved confidence in site-specific predictions. These data demonstrate that while Dorado can detect RNA modifications de novo, its predictions require careful filtering and validation. Our studies of provide a rare benchmark dataset for assessing the ability of transcriptome-wide methods to accurately identify RNA modifications and estimate modification stoichiometry. These findings support the use of orthogonal approaches, including LC-MS/MS and IVT controls, alongside Nanopore sequencing to provide a more reliable and interpretable strategy for studying RNA modification patterns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/729303v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@637a55org.highwire.dtl.DTLVardef@f48272org.highwire.dtl.DTLVardef@a0465aorg.highwire.dtl.DTLVardef@6af045_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Bacterial Schlafens mediate anti-phage defense

Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here, we show that prokaryotic Schlafen nucleases (pSlfns) are widespread antiviral effectors that protect bacteria from phages. These nucleases are fused to diverse protein domains that sense phage infection. We focus on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrate that it recognizes T5-like phage tail assembly chaperones and cleaves both bacterial and viral tRNA, triggering abortive infection. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.

molecular biology↗

bioRxiv2023

The diverse evolutionary histories of domesticated metaviral capsid genes in mammals

Selfish genetic elements and their remnants comprise at least half of the human genome. Active transposons duplicate by inserting copies at new sites in a host genome. Following insertion, transposons can acquire mutations that render them inactive; the accrual of additional mutations can render them unrecognizable over time. However, in rare instances, segments of transposons become useful for the host, in a process called gene domestication. Using the first complete human genome assembly and 25 additional vertebrate genomes, we analyzed the evolutionary trajectories and functional potential of genes domesticated from the capsid genes of Metaviridae, a retroviral-like retrotransposon family. Our analysis reveals four families of domesticated capsid genes in placental mammals with varied evolutionary outcomes, ranging from universal retention to lineage-specific duplications or losses and from purifying selection to lineage-specific rapid evolution. The four families of domesticated capsid genes have divergent amino-terminal domains, inherited from four distinct ancestral metaviruses. Structural predictions reveal that many domesticated genes encode a previously unrecognized RNA-binding domain retained in multiple paralogs in mammalian genomes both adjacent to and independent from the capsid domain. Collectively, our study reveals diverse outcomes of domestication of diverse metaviruses, which led to structurally and evolutionarily diverse genes that encode important, but still largely-unknown functions in placental mammals.

bioinformatics↗

bioRxiv2023

CRISPR-based engineering of RNA viruses

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a new recombinant RNA technology with immediate applications for the facile engineering of RNA viruses. One-Sentence SummaryProgrammable CRISPR RNA-guided ribonucleases enable recombinant RNA technology.

molecular biology↗

bioRxiv2022

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

When eukaryotic cells are killed by pathogenic microorganisms, damage-associated and pathogen-associated signals are generated that alert other cells of nearby danger. Bacteria can detect the death of their kin; however, how bacteria make threat assessments of cellular injury is largely unexplored. Here we show that polyamines released by lysed bacteria serve as damage-associated molecules in Pseudomonas aeruginosa. In response to exogenous polyamines, Gac/Rsm and cyclic-di-GMP signaling is activated and intracellular polyamine levels increase. In the absence of a threat, polyamines are catabolized, and intracellular polyamines return to basal levels, but cells infected by bacteriophage increase and maintain intracellular polyamine levels, which inhibits phage replication. Phage species not inhibited by polyamines did not trigger polyamine accumulation by P. aeruginosa, suggesting polyamine accumulation and metabolism are targets in the phage-host arms-race. Our results suggest that like eukaryotic cells, bacteria can differentiate damage-associated and pathogen-associated signals to make threat assessments of cellular injury. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/486733v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f69334org.highwire.dtl.DTLVardef@13a4422org.highwire.dtl.DTLVardef@4f3edeorg.highwire.dtl.DTLVardef@a86c43_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

The rise and fall of SARS-CoV-2 variants and the emergence of competing Omicron lineages

In late December of 2019, high throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we analyze a subset (n=83,204) of all publicly available SARS-CoV-2 genomes (n=~5.6 million) that were randomly selected, but equally distributed over the course of the pandemic. We plot the emergence and extinction of new variants of concern (VOCs) over time and show how this corresponds to the ongoing accumulation of mutations in SARS-CoV-2 genomes and individual proteins. While the accumulation of mutations generally follows a linear regression, non-synonymous mutations are significantly greater in Omicron viruses than in previous variants-especially in the spike and nucleoproteins-and these differences are more pronounced in a recently identified sub-lineage (BA.2) of Omicron. ImportanceOmicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike and nucleoproteins have accumulated the most mutations in Omicron variants, but that the accessory proteins of SARS-CoV-2 sequences are changing most rapidly relative to their size. Collectively, this "Observation" provides a concise overview of SARS-CoV-2 evolution, reveals mutational differences between two Omicron lineages, and highlights changes in the SARS-CoV-2 proteome that have been under reported.

evolutionary biology↗

bioRxiv2021

The rise and fall of SARS-CoV-2 variants and the mutational profile of Omicron

In late December of 2019, high throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we analyze a subset (n=87,032) of all publicly available SARS-CoV-2 genomes (n=[~]5.6 million) that were randomly selected, but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron viruses are significantly greater than those in previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike protein, while 25 other viral proteins-- including those involved in SARS-CoV-2 replication--are highly conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered. ImportanceOmicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike protein is a hotspot for viral evolution in all variants, suggesting that existing vaccines and diagnostics that target this protein may become less effective against Omicron and that our therapeutic and public health strategies will have to evolve along with the virus.

evolutionary biology↗