Microbiology & Cell Biology
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Item Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection(Proceedings of the National Academy of Sciences, 2023-02) de Mattos, Camilla D.; Faith, Dominick R.; Nemudryi, Artem; Schmidt, Amelia K.; Bublitz, DeAnna C.; Hammond, Lauren R.; Kinnersley, Margie; Schwartzkopf, Caleb M.; Robinson, Autumn J.; Joyce, Alex; Michaels, Lia A.; Brzozowski, Robert S.; Coluccio, Alison; Xing, Denghui David; Uchiyama, Jumpei; Jennings, Laura K.; Eswara, Prahathees; Wiedenheft, Blake; Secor, Patrick R.Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.Item Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription(Cold Spring Harbor Laboratory, 2024-06) Snyder, Laura R.; Kilde, Ingrid; Nemudryi, Artem; Wiedenheft, Blake; Koutmos, Markos; Koutmou, Kristin S.SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA life cycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or antiviral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N6-methyladenosine and 2′-O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.Item Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells(American Association for the Advancement of Science, 2024) Nemudraia, Anna; Nemudryi, Artem; Wiedenheft, BlakeGenome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.Item The Diverse Evolutionary Histories of Domesticated Metaviral Capsid Genes in Mammals(Oxford University Press, 2024-04) Henriques, William S.; Young, Janet M.; Nemudryi, Artem; Nemudraia, AnnaSelfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae, giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals.