About

Ralph Kleiner is an Associate Professor of Chemistry at Princeton University, specializing in the study of cellular RNA function and regulation through chemical biology. His research focuses on understanding the post-transcriptional regulation of RNA by interactions with RNA-binding proteins (RBPs) and chemical modifications known as epitranscriptomic modifications. His lab develops chemical tools to measure various aspects of cellular RNA, including synthesis, turnover, localization, and structure, employing a combination of chemical and biological methods across in vitro and cellular systems. Kleiner's work aims to elucidate the biochemical and biological functions of RBPs, which control all aspects of the RNA lifecycle and are implicated in human diseases. His research also investigates RNA modifications, which diversify RNA structures and influence gene expression regulation. He develops novel chemical approaches to discover RNA-modifying enzymes and characterize their substrates, contributing to the understanding of how RNA modifications impact health and disease. His contributions have been recognized with awards such as the NSF CAREER Award, Sloan Research Fellowship, and the Kavli Fellowship, among others.

Research topics

• Biology
• Chemistry
• Computational biology
• Cell biology
• Biochemistry

Selected publications

• Metabolism of Epigenetic Ribonucleosides Leads to Nucleolar Stress and Cytotoxicity

  ACS Chemical Biology · 2026-03-06

  articleOpen accessSenior authorCorresponding

  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 epigenetic ribonucleoside metabolism in human cells by studying effects on cell growth, quantifying RNA misincorporation 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, consistent with a recent report. Epigenetic ribopyrimidines also exhibit cytotoxicity, dependent on 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.

  PublisherDOI

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

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-18

  articleOpen accessSenior authorCorresponding

  ABSTRACT 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 (cnmn 5 U)-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 (m 5 U) methyltransferases. We use nucleoside and oligonucleotide mass spectrometry to establish that YfjO (which we rename as RlmS) installs m 5 U620 in the 23S rRNA (U576 in E. coli ), a modification specific to the B. subtilis ribosome. We characterize Δ 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 .

  PublisherDOI

• Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

  Nature Communications · 2025-01-26 · 19 citations

  articleOpen access

  While all native tRNAs undergo extensive post-transcriptional modifications as a mechanism to regulate gene expression, mapping these modifications remains challenging. The critical barrier is the difficulty of readthrough of modifications by reverse transcriptases (RTs). Here we use Induro-a new group-II intron-encoded RT-to map and quantify genome-wide tRNA modifications in Induro-tRNAseq. We show that Induro progressively increases readthrough over time by selectively overcoming RT stops without altering the misincorporation frequency. In a parallel analysis of Induro vs. a related RT, we provide comparative datasets to facilitate the prediction of each modification. We assess tRNA modifications across five human cell lines and three mouse tissues and show that, while the landscape of modifications is highly variable throughout the tRNA sequence framework, it is stabilized for modifications that are required for reading of the genetic code. The coordinated changes have fundamental importance for development of tRNA modifications in protein homeostasis.

  PublisherOA PDFDOI

• Abstract 1068 Targeting Pyrimidine Biosynthesis in Entamoeba histolytica: Structural and Biochemical Insights into UMP-CMP Kinase

  Journal of Biological Chemistry · 2025-05-01

  articleOpen access

  Amoebiasis, caused by Entamoeba histolytica, remains a global health burden, affecting over 30 million individuals annually. Current treatments face growing challenges due to emerging drug resistance, necessitating the development of new therapeutic targets. In this study, we characterize uridine monophosphate-cytidine monophosphate kinase (EhUMPK), an enzyme essential for nucleotide biosynthesis in E. histolytica. UMPKs are involved in the phosphorylation of UMP and CMP, which are crucial for nucleic acid synthesis, making them attractive targets for drug development.

  PublisherOA PDFDOI

• Dynamic Regulation of 5-Formylcytidine on tRNA

  ACS Chemical Biology · 2025-03-13

  articleSenior authorCorresponding

  Post-transcriptional modifications on RNA play an important role in biological processes, but we lack an understanding of the molecular mechanisms underlying the function of many modifications. Here we characterize the distribution and dynamic regulation of 5-formylcytidine (f5C), a modification primarily found on tRNAs, across different cell lines, mouse tissues, and in response to environmental stress. We identify perturbation in bulk f5C levels using nucleoside LC-MS and quantify individual modification stoichiometry at the wobble base of mt-tRNA-Met and tRNA-Leu-CAA using nucleotide resolution f5C sequencing technology. Our studies show that f5C modifications on tRNAs are dynamic, and responsive to fluctuations in cellular iron levels and O2 concentration. Further, we show using a translation reporter assay that decoding of Leu UUA codons is impaired in cells lacking f5C, implicating f5C(m)34 on tRNA-Leu-CAA in wobble decoding. Together, our work illuminates dynamic epitranscriptomic mechanisms regulating protein translation in response to environment.

  PublisherDOI

• Amino acid changes in two viral proteins drive attenuation of the yellow fever 17D vaccine

  Nature Microbiology · 2025-07-08 · 8 citations

  articleOpen access

  The live-attenuated yellow fever 17D vaccine strain differs genetically only minimally from its virulent parent. However, it remains unclear which sequence differences lead to virulence or attenuation. Here we demonstrate, using SHAPE-MaP, that these mutations do not induce global RNA structure changes and show that protein sequence mutations are mostly responsible for the phenotypic differences between 17D and virulent YFV. Using a highly modular, combinatorial genetic approach, we identified key mutations in the envelope (E) and non-structural 2A (NS2A) proteins that increase 17D’s ability to spread and enhance host antiviral responses. Introducing these mutations into infectious clones of virulent YFV genomes results in viral attenuation in vitro and in two mouse models. Collectively, our results define the genetic basis for 17D attenuation and highlight a potentially general approach for creating live-attenuated vaccines by introducing mutations resulting in similar phenotypic changes in other pathogenic viruses. Amino acid changes in the envelope and non-structural 2A protein attenuate the virulent yellow fever virus (YFV) and enhance host antiviral responses upon infection in vivo. These traits explain the potency of the YFV vaccine.

  PublisherOA PDFDOI

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

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-12

  preprintOpen accessSenior authorCorresponding

  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.

  PublisherOA PDFDOI

• Metabolism of epigenetic ribonucleosides leads to nucleolar stress and cytotoxicity

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-13

  preprintSenior authorCorresponding

  ABSTRACT 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 N 6 -modified adenosines (i.e. i 6 A) exhibit high levels of cytotoxicity and RNA misincorporation, whereas cells dramatically restrict the misincorporation of small N 6 -modified adenosines (i.e. m 6 A), 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.

  PublisherDOI

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

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-17

  preprintOpen access

  Abstract Modification of RNA with N 6 -methyladenosine (m 6 A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m 6 A, 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 m 6 A writers Mettl3 and Mettl14 , nor the m 6 A 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.

  PublisherOA PDFDOI

• The impact of epitranscriptomic modifications on liver disease

  Trends in Endocrinology and Metabolism · 2024-01-11 · 11 citations

  review
  PublisherDOI

Recent grants

• Chemical Approaches to Illuminate the Epitranscriptome

  NIH · $1.6M · 2019–2025

• CAREER: A Chemoproteomic Strategy to Decipher Epitranscriptomic Pyrimidine Modifications

  NSF · $1.0M · 2019–2024

Frequent coauthors

• David R. Liu

  Howard Hughes Medical Institute

  20 shared

• A. Emilia Arguello

  University of Cambridge

  10 shared

• Adam Claridge‐Chang

  Duke-NUS Medical School

  9 shared

• Danyang Wang

  6 shared

• Nathan J. Yu

  Princeton University

  6 shared

• Christoph E. Dumelin

  5 shared

• Brian Joseph

  Columbia University

  5 shared

• Michael E. Birnbaum

  Allen Institute

  5 shared

Labs

• Kleiner LabPI

Awards & honors

• International Chemical Biology Society (ICBS) Young Chemical…
• Kavli Fellow (2023)
• NSF CAREER Award (2019)
• Sloan Research Fellowship (2019)
• Sidney Kimmel Foundation Scholar Award (2017)