Schwartz Lab

Cracking the epitranscriptome

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Research

Our lab studies the fundamental building blocks of RNA, the genetic material within our cells. RNA, like DNA, is typically thought to comprise only four building blocks. Yet, this view is oversimplified. Following their formation, the building blocks of RNA can be chemically modified in >170 different ways, giving rise to the ‘epitranscriptome’. These chemical modifications play critical roles in regulating the RNA life-cycle and in modulating cellular and physiological responses. RNA modifications are also of intense interest from applied perspectives, and were key to the success of the SARS-Cov2 mRNA vaccines. Our lab seeks to understand the roles played by RNA modifications in diverse natural contexts, and to harness this knowledge towards the development of improved RNA therapeutics.

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Reading RNA modifications

Understanding RNA modifications

Writing RNA modifications / RNA therapeutics

News

October 22, 2025

Pan-Mod-seq paper is published in Cell journal

September 27, 2024

David’s paper regarding semi-passive shaping of m6A dynamics by mRNA metabolism was published

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Selected Publications

Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome

Garcia-Campos M. A., Georgeson J., Nir R., Reichelt R., Fluke K. A., Matzov D., Iyer V., Burkhart B. W., Lui L., Kustanovich A., Grünberger F., Thalalla-Gamage S., Manage S. H., Gerovac M., Alexandre N., Nobe Y., Nowak J. S., Perera M., Apostle A., Fang S., Glatt S., Jona G., Ferreira-Cerca S., Vogel J., Taoka M., Meier J. L., Westhof E., Santangelo T. J., Grohmann D., Shalev-Benami M. & Schwartz S. (2025) Cell.

Summary Ribosomal RNA (rRNA) constitutes the core of ribosomes and is extensively chemically modified. Technical challenges have precluded systematically dissecting rRNA modifications and their dynamics. We develop Pan-Mod-seq, permitting inference of 16 distinct modifications across dozens of samples in parallel. We applied Pan-Mod-seq to RNA from 14 species spanning all domains of life, cultured under highly diverse conditions. While dynamic modifications are rare in mesophiles, in extreme hyperthermophiles, ∼50% of modifications are dynamic. We dissect the biogenesis and function of a conserved module of tandem m5C-ac4C modifications, co-induced at high temperatures, via enzymes intrinsically regulated by temperature and required for growth at higher temperatures. Cryo-electron microscopy (cryo-EM) structures of ribosomes from wild-type (WT) and enzyme-deficient archaea reveal recurrent molecular interactions through which they confer structural stability, and biophysical studies demonstrate their synergistic thermostabilizing role. Our findings systematically dissect rRNA modification plasticity and pave the way for surveying the rRNA epitranscriptome in health and disease.

Exclusion of m6A from splice-site proximal regions by the exon junction complex dictates m6A topologies and mRNA stability

Uzonyi A., Dierks D., Nir R., Kwon O. S., Toth U., Barbosa I., Burel C., Brandis A., Rossmanith W., Le Hir H., Slobodin B. & Schwartz S. (2023) Molecular Cell. 83, 2, p. 237-251.e7

Accepted version

N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we propose that m6A deposition is not selective. Instead, it is exclusion based: m6A consensus motifs are methylated by default, unless they are within a window of ∼100 nt from a splice junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture, allows in silico recapitulation of experimentally measured m6A profiles. We provide evidence that exclusion from splice junctions is mediated by the exon junction complex (EJC), potentially via physical occlusion, and that previously observed associations between exon-intron architecture and mRNA decay are mechanistically mediated via m6A. Our findings establish a mechanism coupling nuclear mRNA splicing and packaging with the covalent installation of m6A, in turn controlling cytoplasmic decay.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

Sas-Chen A., Thomas J. M., Matzov D., Taoka M., Nance K. D., Nir R., Bryson K. M., Shachar R., Liman G. L. S., Burkhart B. W., Gamage S. T., Nobe Y., Briney C. A., Levy M. J., Fuchs R. T., Robb G. B., Hartmann J., Sharma S., Lin Q., Florens L., Washburn M. P., Isobe T., Santangelo T. J., Shalev-Benami M., Meier J. L. & Schwartz S. (2020) Nature. 583, 7817, p. 638-643

Accepted version

N-4-acetylcytidine (ac(4)C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA(1-3). However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac(4)C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac(4)C at single-nucleotide resolution. In human and yeast mRNAs, ac(4)C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac(4)C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. (AcC)-C-4 is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac(4)C and its potential thermoadaptive role. Our studies quantitatively define the ac(4)C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease(4-6).

Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling

Garcia-Campos M. A., Edelheit S., Toth U., Safra M., Shachar R., Viukov S., Winkler R., Nir R., Lasman L., Brandis A., Hanna J. H., Rossmanith W. & Schwartz S. (2019) Cell. 178, 3, p. 731-747

N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution

Safra M., Sas-Chen A., Nir R., Winkler R., Nachshon A., Bar-Yaacov D., Erlacher M., Rossmanith W., Stern-Ginossar N. & Schwartz S. (2017) Nature. 551, 7679, p. 251-255

Accepted version

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N 1 -methyladenosine (m 1 A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m 1 A at single-nucleotide resolution. Within the cytosol, m 1 A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m 1 A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m 1 A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m 1 A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m 1 A levels was adopted as a potential means of post-transcriptional regulation.

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA

Schwartz S., Bernstein D. A., Mumbach M. R., Jovanovic M., Herbst R. H., Leon-Ricardo B. X., Engreitz J. M., Guttman M., Satija R., Lander E. S., Fink G. & Regev A. (2014) Cell. 159, 1, p. 148-162

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis

Schwartz S., Agarwala S. D., Mumbach M. R., Jovanovic M., Mertins P., Shishkin A., Tabach Y., Mikkelsen T. S., Satija R., Ruvkun G., Carr S. A., Lander E. S., Fink G. R. & Regev A. (2013) Cell. 155, 6, p. X1409-1421

N⁶-methyladenosine (m⁶A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m⁶A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.

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