Biology subjects

Burroughs, M. R.

Publications and source records attributed to Burroughs, M. R..

3 recordsLinked to original sources

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2025

Cytoplasmic localization of PUS7 facilitates a pseudouridine-dependent enhancement of cellular stress tolerance

Pseudouridine ({Psi}) is an abundant post-transcriptional modification found across all classes of RNA. It has been widely speculated that {Psi} inclusion in mRNAs might provide an avenue for cells to control gene expression post-transcriptionally. Here we demonstrate that one of the principal mRNA pseudouridylating enzymes, pseudouridine synthase 7 (PUS7), exhibits a stress-induced accumulation in the cytoplasm of yeast and human epithelial lung cells. Stress-induced and cytoplasmic localization of PUS7 promote {Psi}-incorporation into hundreds of mRNA targets. Furthermore, engineered PUS7 cytoplasmic localization increases cellular fitness under ROS and divalent metal ion stress. Consistent with this, transcripts modified upon PUS7 cytoplasmic localization are enriched within mRNAs encoding proteins involved in divalent metal metabolism and ROS stress pathways. In contrast, tRNA sites modified by PUS7 ({Psi}13 and {Psi}35 are unperturbed). Quantitative proteomics reveal a reshaping of the proteome upon PUS7 relocalization under stress, with proteins involved in metal and ROS homeostasis being particularly sensitive to PUS7 localization. Collectively, our data demonstrate that PUS7 localization alters mRNA pseudouridylation patterns to modulate protein production and enhance cellular fitness.

biochemistry↗

bioRxiv2024

Optimized chemical labeling method for isolation of 8-oxoG-modified RNA, ChLoRox-Seq, identifies mRNAs enriched in oxidation and transcriptome-wide distribution biases of oxidation events post environmental stress

Bulk increases in nucleobase oxidation, most commonly manifesting as the guanine (G) nucleobase modification 8-oxo-7,8-dihydroguanine (8-oxoG), have been linked to several disease pathologies. Elucidating the effects of RNA oxidation on cellular homeostasis is limited by a lack of effective tools for detecting specific regions modified with 8-oxoG. Building on a previously published method for studying 8-oxoG in DNA, we developed ChLoRox-Seq, which works by covalently functionalizing 8-oxoG sites in RNA with biotin. Importantly, this method enables antibody-free enrichment of 8-oxoG-containing RNA fragments for Next Generation Sequencing-based detection of modified regions transcriptome-wide. We demonstrate the high specificity of ChLoRox-Seq for functionalizing 8-oxoG over unmodified nucleobases in RNA and benchmark this specificity to a commonly used antibody-based approach. Key advantages of ChLoRox-Seq include: (1) heightened resolution of RNA oxidation regions (e.g. exon-level) and (2) lower experimental costs. By applying ChLoRox-Seq to mRNA extracted from human lung epithelial cells (BEAS-2B) after exposure to environmentally relevant stress, we observe that 8-oxoG modifications tend to cluster in regions that are G-rich and within mRNA transcripts possessing longer 5 UTR and CDS regions. These findings provide new insight into the complex mechanisms that bias the accumulation of RNA oxidation across the transcriptome. Notably, our analysis suggests the possibility that most mRNA oxidation events are probabilistically driven and that mRNAs that possess more favorable intrinsic properties are prone to incur oxidation events at elevated rates. ChLoRox-Seq can be readily applied in future studies to identify regions of elevated RNA oxidation in any cellular model of interest.

molecular biology↗