Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Biallelic variants in YRDC cause a developmental disorder with progeroid features.

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer.

The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide.

Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.

Functional characterization of the human tRNA methyltransferases TRMT10A and TRMT10B.

The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.

5-methylcytosine modification of an Epstein-Barr virus noncoding RNA decreases its stability.

Many cellular noncoding RNAs contain chemically modified nucleotides that are essential for their function. The Epstein-Barr virus expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2. To examine whether these viral RNAs contain modified nucleotides, we purified native EBERs from EBV-infected cells and performed mass spectrometry analysis. While EBER2 contains no modified nucleotides at stoichiometric amounts, EBER1 was found to carry 5-methylcytosine (m5C) modification. Bisulfite sequencing indicated that a single cytosine of EBER1 is methylated in ∼95% of molecules, and the RNA methyltransferase NSUN2 was identified as the EBER1-specific writer. Intriguingly, ablation of NSUN2 and thus loss of m5C modification resulted in an increase in EBER1 levels. We further found that EBER1 is a substrate for the RNase Angiogenin and cleavage in vivo is dependent on the presence of m5C, providing an explanation as to why loss of m5C increases EBER1 levels. Taken together, our observations indicate that m5C, a modification previously shown for tRNAs to oppose Angiogenin-mediated degradation, can also adversely affect RNA stability.

Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs.

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.

FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA.

Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms.

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.

Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.

AlkAniline-Seq: Profiling of m7 G and m3 C RNA Modifications at Single Nucleotide Resolution.

RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome-wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal-to-noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5'-phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAniline-Seq, enables a deep sequencing-based technology for the simultaneous detection of 7-methylguanosine (m7 G) and 3-methylcytidine (m3 C) in RNA at single nucleotide resolution. As a proof-of-concept, we used AlkAniline-Seq to comprehensively validate known m7 G and m3 C sites in bacterial, yeast, and human cytoplasmic and mitochondrial tRNAs and rRNAs, as well as for identifying previously unmapped positions.

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.

MODOMICS: a database of RNA modification pathways. 2017 update.

MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. In the current database version, we included the following new features and data: extended mass spectrometry and liquid chromatography data for modified nucleosides; links between human tRNA sequences and MINTbase - a framework for the interactive exploration of mitochondrial and nuclear tRNA fragments; new, machine-friendly system of unified abbreviations for modified nucleoside names; sets of modified tRNA sequences for two bacterial species, updated collection of mammalian tRNA modifications, 19 newly identified modified ribonucleosides and 66 functionally characterized proteins involved in RNA modification. Data from MODOMICS have been linked to the RNAcentral database of RNA sequences. MODOMICS is available at http://modomics.genesilico.pl.

Inhibition of PARP1-dependent end-joining contributes to Olaparib-mediated radiosensitization in tumor cells.

Poly-ADP-ribose-polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication-dependent and more profound in HR-deficient tumors. Here, we present a new mode of PARPi-mediated radiosensitization which was observed in four out of six HR-proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication-independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB-repair is switched to PARP1-dependent end-joining (PARP1-EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end-joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1-EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.