Fu Xu

The ability to longitudinally track clonal evolution non-invasively would transform cancer interception strategies, long before late-stage disease when most cancer genomes are analysed. Here, we demonstrate that repeated hair sampling from the same individual followed by exome sequencing enables tracking of somatic evolution in vivo over several months after chemically induced skin carcinogenesis. We found that hair follicles accumulate a higher mutation burden than spatially-matched skin and harbour mutations that spread into surrounding epidermis and persist throughout tumour progression. DNA-damaged follicles enter sustained quiescence that delays replication and repair, creating a reservoir for long-lived mutations. During premalignant progression, carcinogen-associated mutations become enriched as follicular clones expand into adjacent skin. Mutation tracking identified genes that may govern tumour predisposition and initiation, many of which are mutated at high incidence in human cutaneous squamous cell carcinoma cohorts. Hair follicles therefore provide a non-invasive readout to forecast the early development of skin cancer, enabling patient risk stratification.

Dysregulated RNA modifications contribute to cancer progression and therapy resistance, yet the underlying mechanism often remains unknown. Here, we perform CRISPR-based synthetic lethality screens to systematically explore the role of RNA modifications in mediating resistance to antileukemic drugs. We identify the tRNA methyltransferase 5 (TRMT5)–mediated formation of N1-methylguanosine (m1G) in the transfer RNA (tRNA) anticodon loop as essential for mediating drug tolerance to cytarabine and venetoclax (Ven) in acute myeloid leukemia (AML). TRMT5 methylates nearly all mitochondrial and nuclear tRNAs with a guanosine at position 37, but its role in promoting drug tolerance specifically depends on its mitochondrial function. TRMT5 is essential for the dynamic upregulation of mitochondrial messenger RNA translation and oxidative phosphorylation, which are critical for sustaining drug tolerance in leukemia cells. This mitochondrial dependency correlates with therapy outcomes in patients with leukemia: lower expression of electron transport chain genes is linked to poorer outcomes in a cohort of nearly 100 patients with AML undergoing first induction therapy. Finally, we demonstrate that targeted depletion of the TRMT5 protein using a conditional degron, in conjunction with cytarabine and Ven treatment, synergistically induces cell death in drug-tolerant AML cells. Thus, our study reveals TRMT5 as a promising drug target for therapy-resistant leukemia.

Transfer RNAs (tRNAs), the essential adapter molecules in protein translation, undergo various post-transcriptional modifications. These modifications play critical roles in regulating tRNA folding, stability, and codon–anticodon interactions, depending on the modified position. Methods for detecting modified nucleosides in tRNAs include isotopic labeling combined with chromatography, antibody-based techniques, mass spectrometry, and high-throughput sequencing. Among these, high-performance liquid chromatography (HPLC) has been a cornerstone technique for analyzing modified nucleosides for decades. In this protocol, we provide a detailed, streamlined approach to purify and digest tRNAs from yeast cells and analyze the resulting nucleosides using HPLC. By assessing UV absorbance spectra and retention times, modified nucleosides can be reliably quantified with high accuracy. This method offers a simple, fast, and accessible alternative for studying tRNA modifications, especially when advanced technologies are unavailable.

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm⁵), 5-methoxycarbonylmethyl (mcm⁵), or 5-methoxycarbonylhydroxymethyl (mchm⁵) side-chain. The presence of these side-chains allows proper pairing with cognate codons, and they are particularly important in tRNA species where the U₃₄residue is also modified with a 2-thio (s²) group. The first step in the synthesis of the ncm⁵, mcm⁵, and mchm⁵side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles ofSOD1, which encodes the cytosolic Cu, Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeastsod1null mutants are impaired in the formation of 2-thio-modified U₃₄residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNA^Phe. Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutantncs6andncs2alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences ofSOD1/sod1alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.

The translational decoding properties of tRNAs are influenced by post-transcriptional modification of nucleosides in their anticodon region. The Elongator complex promotes the first step in the formation of 5-methoxycarbonylmethyl (mcm⁵), 5-methoxycarbonylhydroxymethyl (mchm⁵), and 5-carbamoylmethyl (ncm⁵) groups on wobble uridine residues in eukaryotic cytosolic tRNAs. Elongator mutants in yeast, worms, plants, mice, and humans not only show a tRNA modification defect, but also a diverse range of additional phenotypes. Even though the phenotypes are almost certainly caused by the reduced functionality of the hypomodified tRNAs in translation, the basis for specific phenotypes is not well understood. Here, we discuss the recent finding that the phenotypes ofSaccharomyces cerevisiaeElongator mutants are modulated by the genetic background. This background-effect is largely due to the allelic variation at theSSD1locus, which encodes an mRNA-binding protein involved in post-transcriptional regulation of gene expression. A nonsensessd1allele is found in several wild-type laboratory strains and the presence of this allele aggravates the stress-induced phenotypes of Elongator mutants. Moreover, other phenotypes, such as the histone acetylation and telomeric gene silencing defects, are dependent on the mutantssd1allele. Thus,SSD1is a genetic modifier of the phenotypes of Elongator-deficient yeast cells.

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm⁵) and 5-carbamoylmethyl (ncm⁵) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm⁵/ncm⁵ groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm⁵/ncm⁵ groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm⁵ and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications. Modified nucleosides in the anticodon region of tRNAs are important for the efficiency and fidelity of translation. The Elongator complex promotes formation of several related modified uridine residues at the wobble position of eukaryotic tRNAs. In yeast, plants, worms, mice and humans, mutations in genes for Elongator subunits lead to a wide variety of different phenotypes. Here, we show that the genetic background modulates the phenotypic consequences of the inactivation of budding yeast Elongator. This background effect is largely a consequence of a polymorphism at the SSD1 locus, encoding a RNA binding protein that influences translation, stability and/or localization of mRNAs. We show that several phenotypes reported for yeast Elongator mutants are either significantly stronger or only detectable in strains harboring a mutant ssd1 allele. Thus, SSD1 is a suppressor of the phenotypes induced by the hypomodification of tRNAs.

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm ⁵ ) and 5-carbamoylmethyl (ncm ⁵ ) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm ⁵ /ncm ⁵ groups in tRNAs, but also a wide variety of phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm ⁵ /ncm ⁵ groups in Saccharomyces cerevisiae . We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm ⁵ and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications. Author Summary: Modified nucleosides in the anticodon region of tRNAs are important for the efficiency and fidelity of translation. The Elongator complex promotes formation of several related modified uridine residues at the wobble position of eukaryotic tRNAs. In yeast, plants, worms, mice and humans, mutations in genes for Elongator subunits lead to a wide variety of different phenotypes. Here, we show that the genetic background modulates the phenotypic consequences of the inactivation of budding yeast Elongator. This background effect is largely a consequence of a polymorphism at the SSD1 locus, encoding a RNA binding protein that influences translation, stability and/or localization of mRNAs. We show that several phenotypes reported for yeast Elongator mutants are either significantly stronger or only detectable in strains harboring a mutant ssd1 allele. Thus, SSD1 is a suppressor of the phenotypes induced by the hypomodification of tRNAs.

Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U34 and their influence on Elongator activity.