October 17, 2025Open Access

Profiling of RNA 8-oxoG marks in Escherichia coli identifies critical intrinsic characteristics that contribute to 8-oxoG accumulation in bacteria

MBMatthew R. BurroughsThe University of Texas at Austin BQBlanca I. Quiñones-DíazThe University of Texas at Austin LCLydia M. ContrerasThe University of Texas at Austin

Key Points

Identifying intrinsic RNA characteristics significantly influences 8-oxoG accumulation in bacteria, aiding in understanding RNA modifications.
Assessment using RNA immunoprecipitation sequencing reveals correlation between 8-oxoG enrichment and factors like CDS length and nucleotide composition.
The constructed multiple linear regression model successfully predicts RNA 8-oxoG levels across various bacterial species with differing oxidative stress tolerances.
These findings highlight the importance of understanding the connection between RNA oxidation and cellular homeostasis in diverse bacterial species.

Abstract

Reactive oxygen species (ROS) are environmentally ubiquitous and known to have pervasive impacts on cellular homeostasis. RNA is vulnerable to oxidative chemical alterations from a variety of endogenous and exogenous sources. The most common chemical modification resulting from ROS exposure to RNA is 8-oxo-7,8-dihydroguanine (8-oxoG)--an oxidized form of the canonical guanine (G) nucleobase. While 8-oxoG modifications are known to impact mRNA processing, understanding the broader biological impact of 8-oxoG requires knowledge of how these modifications accumulate. In this work, we assessed the disparate enrichment of 8-oxoG modifications within RNAs in the E. coli transcriptome using an RNA Immunoprecipitation Sequencing technique with a high-affinity 8-oxoG antibody (8-oxoG-RIP-Seq). Our investigation of the RNA 8-oxoG enrichment landscape uncovered several intrinsic RNA characteristics that correlate with 8-oxoG enrichment. These findings suggest intrinsic characteristics of RNA, most notably relative abundance, CDS length, and G nucleotide composition, significantly influence RNA 8-oxoG accumulation. We harnessed these intrinsic characteristics to construct a simple multiple linear regression model that predicts RNA 8-oxoG accumulation, which we validated in E. coli. This model was subsequently applied to predict 8-oxoG enriched RNA species in four other bacterial species spanning a wide range of oxidative stress tolerances; these predictions suggest that 8-oxoG accumulation is largely species dependent, with limited overlap in RNAs and functional pathways that are more susceptible to elevated levels of 8-oxoG accumulation. Overall, these findings better inform understanding of RNA 8-oxoG patterns in bacteria and have broader impacts towards advancing knowledge of the connection between RNA oxidation and cellular homeostasis.

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