Structural basis of translation inhibition by a valine tRNA-derived fragment.

Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Haloferax volcanii Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of Sulfolobus acidocaldarius ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.

The complete mitochondrial genome of Chibiraga houshuaii (Lepidoptera, Limacodidae) and its phylogenetic implications.

Chibiraga is a mall East Asian genus in the family Limacodidae (slug-moths). The latter includes many agricultural pests. Mitochondrial genome analysis is an important tool for studying insect molecular identification and phylogenetics. However, there are very few mitogenome sequences available for Limacodidae species, and none for the genus Chibiraga at all. To explore the mitogenome features of Chibiraga and verify its phylogenetic position, the complete mitogenome of Chibiraga houshuaii was sequenced and annotated. The complete 15,487 bp genome encoded 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (CR). Most of the PCGs had typical ATN start codons and terminated with TAA or a single T residue. UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met) and AAU (Asn) were the five most frequently used codons. All tRNAs were folded into cloverleaf secondary structure, except for trnS1, which lacked the DHU arm. Phylogenetic analyses within the superfamily Zygaenoidea were performed based on multiple datasets from mitochondrial genes. The results showed that the families Phaudidae, Limacodidae and Zygaenidae were respectively recovered as monophyly; C. houshuaii was clustered in a clade with nettle type larvae in Limacodidae.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Biosynthesis and function of 7-deazaguanine derivatives in bacteria and phages.

SUMMARYDeazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life.

Fungi of the order Mucorales express a "sealing-only" tRNA ligase.

Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.

The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes.

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

Cryo-EM structure of the inactive ribosome complex accumulated in chick embryo cells in cold-stress conditions.

Here, we present the high-resolution structure of the Gallus gallus 80S ribosome obtained from cold-treated chicken embryos. The translationally inactive ribosome complex contains elongation factor eEF2 with GDP, SERPINE1 mRNA binding protein 1 (SERBP1) and deacylated tRNA in the P/E position, showing common features with complexes already described in mammals. Modeling of most expansion segments of G. gallus 28S ribosomal RNA allowed us to identify specific features in their structural organization and to describe areas where a marked difference between mammalian and avian ribosomes could shed light on the evolution of the expansion segments. This study provides the first structure of an avian ribosome, establishing a model for future structural and functional studies on the translational machinery in Aves.

Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

Structural and functional insights into tRNA recognition by human tRNA guanine transglycosylase.

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAsAsp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

An Atlas of the base inter-RNA stacks involved in bacterial translation.

Nucleobase-specific noncovalent interactions play a crucial role in translation. Herein, we provide a comprehensive analysis of the stacks between different RNA components in the crystal structures of the bacterial ribosome caught at different translation stages. Analysis of tRNA||rRNA stacks reveals distinct behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies-rare for inter-chain RNA interactions-with higher average interaction energies than A or P-site stacks. This suggests that stacking may be essential for stabilizing tRNA progression through the E-site. Additionally, mRNA||rRNA stacks reveal other geometries, which depend on the tRNA binding site, whereas 16S rRNA||23S rRNA stacks highlight the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacks exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, both A and E-sites display a more diverse distribution of inter-RNA stacks compared to the P-site. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are likely invoked to compensate and support the integrity and dynamics of translation.

mRNA reading frame maintenance during eukaryotic ribosome translocation.

One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

Insights into the Mechanism of Installation of 5-Carboxymethylaminomethyl Uridine Hypermodification by tRNA-Modifying Enzymes MnmE and MnmG.

The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm5) or a 5-taurinomethyl (τm5) group onto wobble uridines of several tRNA species. The Escherichia coli MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH2THF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm5 in a reaction that also requires hydrolysis of GTP. In this letter, we investigated key steps of the MnmEG reaction using a combination of biochemical techniques. We show multiple lines of evidence supporting flavin-iminium FADH[N5═CH2]+ as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N5═CD2]+ analogue, the intermediacy of the FAD in the transfer of the methylene group from CH2THF to the C5 position of U34 was unambiguously demonstrated. Further, MnmEG reactions containing the deuterated flavin-iminium intermediate and alternate nucleophiles such as taurine and ammonia also led to the formation of the anticipated U34-modified tRNAs, showing FAD[N5═CH2]+ as the universal intermediate for all MnmEG homologues. Additionally, an RNA-protein complex stable to urea-denaturing polyacrylamide gel electrophoresis was identified. Studies involving a series of nuclease (RNase T1) and protease (trypsin) digestions along with reverse transcription experiments suggest that the complex may be noncovalent. While the conserved MnmG cysteine C47 and C277 mutant variants were shown to reduce FAD, they were unable to promote the modified tRNA formation. Overall, this study provides critical insights into the biochemical mechanism underlying tRNA modification by the MnmEG.

The New Mitochondrial Genome of Hemiculterella wui (Cypriniformes, Xenocyprididae): Sequence, Structure, and Phylogenetic Analyses.

Hemiculterella wui is an endemic small freshwater fish, distributed in the Pearl River system and Qiantang River, China. In this study, we identified and annotated the complete mitochondrial genome sequence of H. wui. The mitochondrial genome was 16,619 bp in length and contained 13 protein coding genes (PCGs), two rRNA genes, 22 tRNA genes, and one control region. The nucleotide composition of the mitochondrial genome was 29.9% A, 25.3% T, 27.4% C, and 17.5% G, respectively. Most PCGs used the ATG start codon, except COI and ATPase 8 started with the GTG start codon. Five PCGs used the TAA termination codon and ATPase 8 ended with the TAG stop codon, and the remaining seven genes used two incomplete stop codons (T and TA). Most of the tRNA genes showed classical cloverleaf secondary structures, except that tRNASer(AGY) lacked the dihydrouracil loop. The average Ka/Ks value of the ATPase 8 gene was the highest, while the average Ka/Ks value of the COI gene was the lowest. Phylogenetic analyses showed that H. wui has a very close relationship with Pseudohemiculter dispar and H. sauvagei. This study will provide a valuable basis for further studies of taxonomy and phylogenetic analyses in H. wui and Xenocyprididae.

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2.

5-Methylcytosine (m5 C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m5 C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.

Synthesis and Structure Elucidation of Glutamyl-Queuosine.

Queuosine is one of the most complex hypermodified RNA nucleosides found in the Wobble position of tRNAs. In addition to Queuosine itself, several further modified derivatives are known, where the cyclopentene ring structure is additionally modified by a galactosyl-, a mannosyl-, or a glutamyl-residue. While sugar-modified Queuosine derivatives are found in the tRNAs of vertebrates, glutamylated Queuosine (gluQ) is only known in bacteria. The exact structure of gluQ, particularly with respect to how and where the glutamyl side chain is connected to the Queuosine cyclopentene side chain, is unknown. Here we report the first synthesis of gluQ and, using UHPLC-MS-coinjection and NMR studies, we show that the isolated natural gluQ is the α-allyl-connected gluQ compound.