Biology subjects

Fleming, A. M.

Publications and source records attributed to Fleming, A. M..

11 recordsLinked to original sources

bioRxiv2026

Single-molecule nanopore sequencing reveals spatial coordination of rRNA modifications in human ribosomes

Ribosomal RNA has a high density of epitranscriptomic modifications essential for faithful translation, and their levels vary across cell types and in disease. Typically, rRNA modifications are quantified in bulk, and therefore, coordination on individual RNA molecules has remained poorly understood. Herein, modification-aware, single-molecule nanopore sequencing enabled detection of the co-occurrence of rRNA modifications on individual transcripts, resolving coordination invisible to ensemble methods. An analytical framework was established that separates co-occurrence from read-quality, false-positive, calling-artifact, near-saturation, and global modification-level confounds, using human rRNAs from HEK293T cells as the test dataset. The method was applied to four human cell lines to find that co-occurrence is not generally explained by a shared small nucleolar RNA (snoRNA) guide; instead, coordinated modifications cluster locally, within [~]100 nucleotides and 35-40 angstroms in the folded ribosome. For one shared-guide pair, coordination increased as guide levels fell across cell lines, possibly indicating an all-or-nothing mode of modification per molecule under limiting guide availability. Further, the results revealed rRNA heterogeneity between the cells in overall modification levels. Finally, levofloxacin remodeled specific modification sites and their local co-occurrence networks, showing that the approach can reveal small-molecule perturbation of rRNA modification networks.

biochemistry↗

bioRxiv2025

Nanopore sequencing reveals operon-specific ribosome remodeling accompanying naphthyridone resistance in Staphylococcus aureus

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore sequencing identified a single 23S rRNA mutation (T1732C) confined to one of the six rRNA operons (operon 2), which uniquely contains nine tRNA genes. RNA direct nanopore sequencing generated a comprehensive and updated rRNA modification map for S. aureus and revealed extensive remodeling of rRNA modifications in the resistant strain. Differentially incorporated modifications included pseudouridine, dihydrouridine, and 5-hydroxycytidine at functionally relevant positions within the ribosome. Upon mapping these epitranscriptomic changes, we noted they were operon specific. This likely gives rise to ribosome heterogeneity with potential for selective translation of stress-response genes that favor resistance. Collectively, these findings establish nanopore sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations, providing molecular insight into how bacteria evolve resistance to antibiotics.

biochemistry↗

bioRxiv2025

Ribosomal RNA tentacles are targets of free radical damage in mammalian cells during oxidative and inflammatory stress

Chemical damage to ribosomal RNA (rRNA) during oxidative or inflammatory stress can impact protein synthesis. Human cells were exposed to a H2O2 titration series to induce oxidative stress or to tumor necrosis factor- to induce inflammation over a time course followed by RNA direct nanopore sequencing of cytosolic and mitochondrial rRNAs. The guanosine (G) oxidation sites and deamination of adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) lesion sites were revealed by changes in the base-called data. Both stressors induced G oxidation in cytosolic rRNA, whereas mitochondrial rRNA was less oxidatively modified. Nitrosative stress generated during inflammation resulted in deamination lesions in rRNAs in both compartments. Inspection of highly modified sites showed the GC-rich tentacles in the 28S rRNA sequence were hotspots for G oxidation and C deamination in the cytosolic ribosome. Outside of tentacles, lesions were generally found on nucleotides on the ribosome surface exposed to solvent, where diffusible reactive species exist. The minimalist structure of the mitochondrial ribosome compared to the cytosolic ribosome alters the reaction patterns observed to target nucleotides on the surface or in functionally relevant regions. These patterns support the hypothesis that tentacles in cytosolic ribosomes direct reactive oxygen and nitrogen species away from the catalytic core to maintain ribosome activity during stress, while the mitochondrial ribosome is damaged in regions that can deactivate protein synthesis. The results provide molecular insight into metabolic dysfunction during oxidative and inflammatory stress and suggest a new function for the GC-rich tentacles that have evolved in mammalian cells. Significance StatementInfection and injury trigger a cellular inflammatory response resulting in the release of free radical species capable of DNA and RNA damage. We used RNA direct nanopore sequencing to map oxidized guanosine sites in human ribosomal RNA via base-calling error analysis. Reactive nitrogen species derived from peroxynitrite result in deamination reactions, principally cytidine to uridine and adenosine to inosine, which can be directly read by nanopore sequencing. Importantly, cytosolic ribosomes behave very differently than mitochondrial ribosomes; the latter rRNA is somewhat protected from G oxidation by high levels of bicarbonate as well as a protein-coated ribosome structure. In contrast, cytosolic rRNA tentacles are hotspots for both G oxidation and C and A deamination, which might explain their evolved function.

biochemistry↗

bioRxiv2024

CO2 protects cells from iron-Fenton oxidative DNA damage in E. coli and humans

Whereas hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological levels of bicarbonate. Analysis of the metabolome, transcriptome and, in human cells, the nuclear genome shows a consistent buffering of H2O2-induced oxidative stress leading to one common pathway, namely guanine oxidation. Particularly revealing are nanopore-based studies of direct RNA sequencing of cytosolic and mitochondrial ribosomal RNA along with glycosylase-dependent qPCR studies of oxidative DNA damage in telomeres. The focusing of oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes and their involvement in gene regulation in response to oxidative stress.

biochemistry↗

bioRxiv2024

The Apurinic/Apyrimidinic Endodeoxyribonuclease 1 is an RNA G-quadruplex binding protein and regulates miR-92b expression in cancer cells

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as a DNA repair enzyme, unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized- or abasic-miRNAs during oxidative stress conditions. Furthermore, in recent years several non-canonical pathways of miRNA biogenesis have been described, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. In this study, we show that several miRNA precursors, dysregulated upon APE1-depletion, contain an rG4 motif and that their corresponding target genes are upregulated after APE1-depletion. We also show, both by in vitro assays and by using a HeLa cell model, that APE1 can bind and regulate the folding of an rG4 structure contained in pre-miR92b, with a mechanism strictly dependent on critical lysine residues present in the N-terminal disordered region. Furthermore, APE1 depletion in HeLa cells alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Lastly, bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings for cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver cancer, as suggested by their involvement in several cancer-related pathways. Significance StatementWe highlight an undescribed non-canonical role of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) in the context of RNA G-quadruplexes (rG4), specifically in the alternative pathway of miRNA maturation of guanosine-rich miRNA precursors. Specifically, APE1 binds these structures and modulates their folding, mainly through its N-terminal region and some residues in its catalytic domain. Moreover, we showed an interesting new role of APE1 in regulating the shuttling and accumulation of miR-92b between the nuclear and cytosolic compartments, opening new perspectives on how APE1 may exercise its role in the miRNA maturation pathway and function. Moreover, APE1-depleted dysregulated miRNAs with rG4 motifs in their precursors have significant prognostic value in lung, cervical, and liver tumors, suggesting potential targets for cancer therapy.

molecular biology↗

bioRxiv2024

DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context

Molecular details for DNA damage impact on the folding of potential G-quadruplex sequences (PQS) to non-canonical DNA structures that are involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, referred to as a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg2+ ions and the G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1H-NMR and CD analyses confirmed that isothermal folding of the VEGF-DGD constructs yielded spectral signatures that suggest formation of G4 motifs, and demonstrated a folding dependency with the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/576387v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@19c5e3eorg.highwire.dtl.DTLVardef@ef72b7org.highwire.dtl.DTLVardef@5475e4org.highwire.dtl.DTLVardef@107d836_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Nanopore sequencing for the 17 modification types in 36 locations in E. coli ribosomal RNA enables monitoring of stress-dependent changes

Escherichia coli possess the 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Direct RNA nanopore sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to non-modified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knock-out E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modifications signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. In the 16S rRNA, there resides an m4Cm modification at site 1402 that decreased with both stressors. Using helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to the seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

biochemistry↗

bioRxiv2022

Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine ({Psi}), or N1-methylpseudouridine (m1{Psi}) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, {Psi}, or m1{Psi} as they translocated through the helicase and nanopore proteins identified diagnostic signatures for {Psi} and m1{Psi}; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for {Psi} and m1{Psi} enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with {Psi}TP or m1{Psi}TP lead to nucleotide selection in all possible adjacent sequence contexts. For UTP vs. {Psi}TP, {Psi}TP was favorably incorporated in singly-modified contexts, while doubly-modified contexts found high yields of {Psi}TP insertion on the 5' side and lower yields on the 3' side. For UTP vs. m1{Psi}TP, UTP was favorably selected except in 5'-XA (X = U or m1{Psi}) where the ratio was determined by their relative NTP concentrations. Experiments with chemically-modified triphosphates and DNA templates designed based on the structure of T7 RNA polymerase provide a model to explain the observations. These results may aid in future efforts that employ IVT to make therapeutic mRNAs with sub-stochiometric amounts of m1{Psi}.

biochemistry↗

bioRxiv2021

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+ and ionic strength

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using {Delta}33 or {Delta}61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA[->]G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/457676v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@91f32borg.highwire.dtl.DTLVardef@1f3a4f6org.highwire.dtl.DTLVardef@d33240org.highwire.dtl.DTLVardef@1882c13_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Nanopore dwell time analysis permits sequencing and conformational assignment of pseudouridine in SARS-CoV-2

Nanopore devices can directly sequence RNA, and the method has the potential to determine locations of epitranscriptomic modifications that have grown in significance because of their roles in cell regulation and stress response. Pseudouridine ({Psi}), the most common modification in RNA, was sequenced with a nanopore system using a protein sensor with a helicase brake in synthetic RNAs with 100% modification at 18 known human pseudouridinylation sites. The new signals were compared to native uridine (U) control strands to characterize base calling and associated errors as well as ion current and dwell time changes. The data point to strong sequence context effects in which {Psi} can easily be detected in some contexts while in others {Psi} yields signals similar to U that would be false negatives in an unknown sample. We identified that the passage of {Psi} through the helicase brake slowed the translocation kinetics compared to U and showed a smaller sequence bias that could permit detection of this modification in RNA. The unique signals from {Psi} relative to U are proposed to reflect the syn-anti conformational flexibility of {Psi} not found in U, and the difference in {pi} stacking between these bases. This observation permitted analysis of SARS-CoV-2 nanopore sequencing data to identify five conserved {Psi} sites on the 3 end of the viral sub-genomic RNAs, and other less conserved {Psi} sites. Using the helicase as a sensor protein in nanopore sequencing experiments enables detection of this modification in a greater number of relevant sequence contexts. The data are discussed concerning their analytical and biological significance.

biochemistry↗

bioRxiv2020

Potential G-quadruplex forming sequences and N6-methyladenosine colocalize at human pre-mRNA intron splice sites

Using bioinformatic analysis of published data, we identify in human mRNA that potential G-quadruplex forming sequences (PQSs) colocalize with the epitranscriptomic modifications N6-methyladenosine (m6A), pseudouridine ({Psi}), and inosine (I). A deeper analysis of the colocalized m6A and PQSs found them intronic in pre-mRNA near 5' and 3' splice sites. The loop lengths and sequence context of the m6A-bearing PQSs found short loops most commonly comprised of A nucleotides. This observation is consistent with literature reports of intronic m6A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m6A and PQSs in pre-mRNA at intron splice junctions suggests that these features could be involved in alternative mRNA splicing. A similar analysis for PQSs around sites of {Psi} installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m6A. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/939116v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@c5fdf3org.highwire.dtl.DTLVardef@a37482org.highwire.dtl.DTLVardef@136280forg.highwire.dtl.DTLVardef@8023ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗