Biology subjects

Kleiner, R. E.

Publications and source records attributed to Kleiner, R. E..

13 recordsLinked to original sources

bioRxiv2026

Activity-based profiling of bacterial RNA-modifying enzymes reveals species-specific 5-methyluridine modification in Bacillus subtilis 23S rRNA

RNA modifications play an important role in biological processes. Mapping the diversity of RNA chemistry and studying the biological function of individual modifications remains an outstanding challenge in many organisms. In particular, RNA modifications remain poorly studied across most bacterial systems. Our group previously developed RNA-mediated activity-based protein profiling (RNABPP), a reactivity-based strategy employing metabolic labeling and quantitative proteomics to profile RNA modification writer enzymes in human cells. Here we adapt this approach to characterize RNA-modifying enzymes in bacteria. We apply metabolic labeling with 5-fluoropyrimidine nucleosides and phase separation-based enrichment of RNA-protein complexes (RNABPP-PS) to profile RNA pyrimidine modifying enzymes in E. coli and B. subtilis. We identify known and putative bacterial pyrimidine C5 methyltransferases, pseudouridine synthases, and dihydrouridine synthases, demonstrating the utility of our approach. Further, we find the carboxymethylaminomethyluridine (cnmn5U)-forming enzyme MnmG (GidA), supporting the existence of a covalent protein-RNA intermediate during the catalytic cycle. Finally, we use RNABPP-PS in B. subtilis to identify YfjO, an uncharacterized protein that is homologous to 5-methyluridine (m5U) methyltransferases. We use nucleoside and oligonucleotide mass spectrometry to establish that YfjO (which we rename as RlmS) installs m5U620 in the 23S rRNA (U576 in E. coli), a modification specific to the B. subtilis ribosome. We characterize {Delta}yfjO B. subtilis which has impaired cell proliferation and protein translation rate compared to WT. Taken together, our study establishes a versatile platform for RNA modifying enzyme discovery and characterization in bacteria and illuminates species-specific rRNA modification chemistry in B. subtilis.

biochemistry↗

bioRxiv2025

Targeted RNA editing by direct delivery of an adenosine deaminase-antisense oligo conjugate

Programmable RNA editing is a promising therapeutic strategy for correcting disease-causing mutations on mRNA, but current approaches rely primarily upon endogenous RNA editing enzymes (i.e. ADARs) that have restricted substrate scope and efficiency. Here, we demonstrate programmable RNA editing with evolved TadA-derived deaminases and 2-methoxyethyl (MOE)-modified antisense oligonucleotides (ASO) to guide site-directed A-to-I editing. In contrast to ADAR enzymes, TadA proteins modify single-stranded RNA (ssRNA). We profile ASO-guided TadA-based editors on endogenous and disease-relevant mRNAs and develop a "bulge-forming" ASO architecture to constrain RNA editing to the target site. Further, we demonstrate that a covalent adenosine deaminase-ASO "RNP" conjugate formed in the test tube and delivered by lipofection achieves targeted and efficient RNA editing with dramatically lower transcriptome-wide off-target editing as compared to ectopically expressed RNA editing enzymes. Taken together, our work expands the scope of programmable RNA editing methods with broad implications for therapeutic modulation of RNA behavior.

biochemistry↗

bioRxiv2025

Metabolism of epigenetic ribonucleosides leads to nucleolar stress and cytotoxicity

Post-transcriptional RNA modifications are ubiquitous in biology, but the fate of epigenetic ribonucleotides after RNA turnover and the consequences of their metabolism and misincorporation into nucleic acids are largely unknown. Here we explore the metabolism of epigenetic ribonucleosides in human cells by studying effects on cell growth, quantifying misincorporation into cellular RNAs and identifying metabolic regulators, and exploring phenotypes associated with cytotoxicity. We find that bulky N6-modified adenosines (i.e. i6A) exhibit high levels of cytotoxicity and RNA misincorporation, whereas cells dramatically restrict the misincorporation of small N6-modified adenosines (i.e. m6A), partly through sanitization by enzymatic deamination. Epigenetic ribopyrimidines also exhibit cytotoxicity, mediated primarily by nucleoside kinase UCK2, but only at much higher concentrations than ribopurines. We further characterize the effects of cytotoxic ribonucleoside metabolism on nucleolar morphology and protein translation. Taken together, our work provides new insights into the metabolism of epigenetic ribonucleosides and mechanisms underlying their cytotoxicity to cells.

biochemistry↗

bioRxiv2024

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

molecular biology↗

bioRxiv2023

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity

Monitoring the dynamic changes of cellular tRNA pools is challenging, due to the extensive post-transcriptional modifications of individual species. The most critical component in tRNAseq is a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. Using our Induro-tRNAseq, simpler and more comprehensive than the best methods to date, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops, without altering the misincorporation frequency. We provide a parallel dataset of the misincorporation profile of Induro relative to the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology of decoding proline codons.

genomics↗

bioRxiv2023

A minimal sequence motif drives selective tRNA dihydrouridylation by hDUS2

The post-transcriptional reduction of uridine to dihydrouridine (D) by dihydrouridine synthase (DUS) enzymes is among the most ubiquitous transformations in RNA biology. D is found at multiple sites in tRNAs and studies in yeast have proposed that each of the four eukaryotic DUS enzymes modifies a different site, however the molecular basis for this exquisite selectivity is unknown and human DUS enzymes have remained largely uncharacterized. Here we investigate the substrate specificity of human dihydrouridine synthase 2 (hDUS2) using mechanism-based crosslinking with 5-bromouridine (5-BrUrd)-modified oligonucleotide probes and in vitro dihydrouridylation assays. We find that hDUS2 modifies U20 in the D loop of diverse tRNA substrates and identify a minimal GU motif within the tRNA tertiary fold required for directing its activity. Further, we use our mechanism-based platform to screen small molecule inhibitors of hDUS2, a potential anti-cancer target. Our work elucidates the principles of substrate modification by a conserved DUS and provides a general platform to studying RNA modifying enzymes with sequence-defined activity-based probes.

biochemistry↗

bioRxiv2023

Cell type-specific translational regulation by Human DUS enzymes

Dihydrouridine is an abundant and conserved modified nucleoside present on tRNA, but characterization and functional studies of modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as an activity-based probe for human DUS enzymes. We map D modifications using RNA-protein crosslinking and chemical transformation and mutational profiling to reveal D modification sites on human tRNAs. Further, we knock out individual DUS genes in two human cell lines to investigate regulation of tRNA expression levels and codon-specific translation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.

biochemistry↗

bioRxiv2023

Transcriptome-wide RNA structure probing with temporal resolution

Reactive small-molecule probes are widely used for RNA structure probing, however current approaches largely measure average RNA transcript dynamics and do not resolve structural differences that occur during folding or transcript maturation. Here, we present SNIPER-seq, an RNA structure probing method relying upon metabolic labeling with 2-aminodeoxycytidine, structure-dependent 2-amino reaction with an aromatic isothiocyanate, and high-throughput RNA sequencing. Our method maps cellular RNA structure transcriptome-wide with temporal resolution enabling determination of transcript age-dependent RNA structural dynamics. We benchmark our approach against known RNA structures and investigate the dynamics of human 5S rRNA during ribosome biogenesis, revealing specific structural changes in 5S rRNA loops that occur over the course of several hours. Taken together, our work sheds light on the maturation and coordinated conformational changes that take place during ribosome biogenesis and provides a general strategy for surveying evolving RNA structural dynamics across the transcriptome.

biochemistry↗

bioRxiv2023

Chemoproteomic profiling of 8-oxoguanosine-sensitive RNA-protein interactions

Cellular nucleic acids are subject to assault by endogenous and exogenous agents that can perturb the flow of genetic information. Oxidative stress leads to the accumulation of 8-oxoguanine (8OG) on DNA and RNA. 8OG lesions on mRNA negatively impact translation, but their effect on global RNA-protein interactions is largely unknown. Here, we apply an RNA chemical proteomics approach to investigate the effect of 8OG on RNA-protein binding. We find proteins that bind preferentially to 8OG-modified RNA, including IGF2BP1-3 and hnRNPD, and proteins that are repelled by 8OG such as RBM4. We characterize these interactions using biochemical and biophysical assays to quantify the effect of 8OG on binding and show that a single 8OG abolishes binding of RBM4 to its preferred CGG-containing substrate. Taken together, our work establishes the molecular consequences of 8OG on cellular RNA-protein binding and provides a framework for interrogating the role of RNA oxidation in biological systems.

biochemistry↗

bioRxiv2023

Oxaliplatin Inhibits RNA Polymerase I via ATM- and ATR-Dependent Signaling Targeted to the Nucleolus

Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mode of action. In particular, emerging evidence indicates that oxaliplatin, a Pt drug used to treat colorectal cancer, kills cells by inducing ribosome biogenesis stress rather than through DNA damage generation, but the underlying mechanism is unknown. Here, we demonstrate that oxaliplatin-induced ribosomal RNA (rRNA) transcriptional silencing and nucleolar stress occur downstream of DNA damage signaling involving ATM and ATR. We show that NBS1 and TOPBP1, two proteins involved in the nucleolar DNA damage response (n-DDR), are recruited to nucleoli upon oxaliplatin treatment. However, we find that rRNA transcriptional inhibition by oxaliplatin does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing it from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct DDR signaling pathway that functions in trans to inhibit Pol I transcription in the nucleolus, demonstrating how nucleolar stress can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity.

molecular biology↗

bioRxiv2022

Profiling dynamic RNA-protein interactions using small molecule-induced RNA editing

RNA binding proteins (RBPs) play an important role in biology and characterizing dynamic RNA-protein interactions in their native context is essential for understanding RBP function. Here, we develop targets of RNA-binding proteins identified by editing induced through dimerization (TRIBE-ID), a facile strategy for identifying and quantifying state-specific RNA-protein interactions based upon rapamycin-mediated chemically induced dimerization and RNA editing. We perform TRIBE-ID with G3BP1, an abundant RBP and core component of stress granules, to study transcriptome-wide G3BP1-RNA interactions during normal conditions and upon oxidative stress-induced liquid-liquid phase separation (LLPS). We quantify editing kinetics in order to infer interaction persistence and show that stress granule formation strengthens preexisting G3BP1-RNA interactions and induces new RNA-protein binding events. Further, we demonstrate that G3BP1 stabilizes its RNA clients in a dose-dependent manner, suggesting that stress granules function as RNA storage depots. Finally, we apply our method to characterize small molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile RNA-protein binding events with temporal control and illuminates the role of LLPS in organizing G3BP1-RNA interactions in the cell.

biochemistry↗

bioRxiv2022

Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems, however approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2 (UCK2). In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with quantitative confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Further, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

biochemistry↗

bioRxiv2021

A chemical method to sequence 5-formylcytosine on RNA

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method to sequence 5-formylcytosine (f5C) modifications on RNA based upon selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and PCR amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

biochemistry↗