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A small cationic probe highly selective for RNA tertiary structure

ABSTRACT: RNA is unique among the large biomolecules due to the intersection of its distinctive electrostatic properties, created by the permanent charge at each phosphate group, coupled with its ability to form diverse three-dimensional tertiary structures. We demonstrate that at rare, but distinctive, sites tertiary folding creates electronegative pockets that preferentially react with a small positively-charged chemical probe, trimethyloxonium (TMO), which alkylates RNA. TMO performs the same chemistry with RNA as the classical uncharged probe, dimethyl sulfate (DMS). Sites preferentially reactive with the TMO cation, relative to the uncharged DMS, are detected efficiently by massively parallel sequencing, at low read depths. These motifs, which we call T-sites, reflect preferential interaction of TMO at an electronegative pocket created by juxtaposition of a reactive nucleobase with non-bridging oxygen atoms, and specifically map to the centers of higher-order structural interactions and functional cores in model RNAs. T-site probing is a facile robust strategy, poised to create broad opportunities to detect and analyze RNA tertiary structures transcriptome-wide.

ORGANISM(S): Geobacillus stearothermophilus Escherichia coli Oceanobacillus iheyensis Orthoflavivirus denguei

PROVIDER: GSE286293 | GEO | 2025/10/29

REPOSITORIES: GEO

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Project description:Background/Objectives: Obese subjects have increased number of enlarged fat cells which are reduced in size but not number in post-obesity. We performed DNA methylation profiling in fat cells with the aim of identifying differentially methylated DNA sites (DMS) linked to adipose hyperplasia (many small fat cells) in post-obesity. Subjects/Methods: Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells from 16 women examined two years after gastric bypass surgery at a post-obese state (BMI 26±2 kg/m2, mean±s.d.) and 14 never-obese women (BMI 25±2 kg/m2). Gene expression was analyzed in subcutaneous adipose tissue from 9 women in each group. In a secondary analysis, we examined DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. Results: The average degree of DNA methylation of all analyzed CpG-sites was lower in fat cells from post-obese as compared to never-obese women (P=0.014). 8,504 CpG sites were differentially methylated in fat cells from post-obese versus never-obese women (false discovery rate 1%). DMS were under-represented in CpG-islands and surrounding shores. The 8,504 DMS mapped to 3,717 unique genes; these genes were over-represented in cell differentiation pathways. Notably, 27% of genes linked to adipogenesis (i.e. 35 of 130) displayed DMS (adjusted P=10−8) in post-obese versus never-obese women. Next, we explored DNA methylation and expression of genes linked to adipogenesis in more detail in adipose tissue samples. DMS annotated to adipogenesis genes were not accompanied by differential gene expression in post-obese compared to never-obese women. In contrast, adipogenesis genes displayed differential DNA methylation accompanied by altered expression in obese women, Conclusions: Global CpG hypomethylation and overrepresentation of DMS in adipogenesis genes in fat cells may contribute to adipose hyperplasia in post-obese women. Post obese=16, Control group=14.

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A small cationic probe highly selective for RNA tertiary structure

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