Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp3U47. While overall protein synthesis is not affected in ΔtrmA and ΔtruB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB.

Fluoropyrimidines trigger decay of hypomodified tRNA in yeast.

Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent. tRNA methylation deficiency caused by loss of Trm4 and Trm8 was previously shown to trigger an RNA quality control mechanism resulting in partial destabilization of hypomodified tRNAValAAC. We demonstrate that, following incorporation into tRNA, fluoropyrimidines strongly enhance degradation of yeast tRNAValAAC lacking Trm4 and Trm8 dependent methylations. At elevated temperature, such effect occurs already in absence of Trm8 alone. Genetic approaches and quantification of tRNA modification levels reveal that enhanced fluoropyrimidine cytotoxicity results from additional, drug induced uridine modification loss and activation of tRNAValAAC decay involving the exonuclease Xrn1. These results suggest that inhibition of tRNA methylation may be exploited to boost therapeutic efficiency of 5-FU and 5-FC.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

The virus responsible for COVID-19 infections is known as SARS-CoV-2. Like all viruses, SARS-CoV-2 carries instructions to make proteins and other molecules that play essential roles in enabling the virus to multiply and spread. Viruses are unable to make these molecules themselves, so they infect cells and trick them into making the molecules and assembling new virus particles on their behalf instead. When SARS-CoV2 infects cells, the host cells are reprogrammed to make chains containing several virus proteins that need to be severed from each other by a virus enzyme, known as Nsp5, to enable the proteins to work properly. Previous studies suggested that Nsp5 may also interact with a human protein known as TRMT1, which helps with the production of new proteins in cells. However, it was not clear how Nsp5 may bind to TRMT1 or how this interaction may affect the host cell. Zhang et al. used biochemical and molecular techniques in human cells to study how Nsp5 interacts with TRMT1. The experiments found that the virus enzyme cuts TRMT1 into fragments that are inactive and are subsequently destroyed by the cells. Moreover, Nsp5 cuts TRMT1 at exactly the same position corresponding to the cleavage sites of the viral proteins. Mutation of the sequence in TRMT1 renders Nsp5 ineffective at cutting the protein. SARS-CoV-2 infection caused TRMT1 levels to decrease inside the cells, in turn, leading to a drop in TRMT1 activity. The virus multiplied less in cells that were unable to produce TRMT1 compared to normal human cells, suggesting that the virus benefits from TRMT1 early during infection, before inactivating it at a later point. These findings suggest that one way SARS-CoV-2 causes disease is by decreasing the levels of a human protein that regulates protein production. In the future, the work of Zhang et al. may provide new markers for detecting infections of SARS-CoV-2 and other similar viruses and guide efforts to make more effective therapies against them.

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.

The epigenetic downregulation of LncGHRLOS mediated by RNA m6A methylase ZCCHC4 promotes colorectal cancer tumorigenesis.

BACKGROUND: m6A modification is currently recognized as a major driver of RNA function that maintains cancer cell homeostasis. Long non-coding (Lnc) RNAs control cell proliferation and play an important role in the occurrence and progression of colorectal cancer (CRC). ZCCHC4 is a newly discovered m6A methyltransferase whose role and mechanism in tumors have not yet been elucidated. METHODS: The EpiQuik m6A RNA methylation kit was used to detect the level of total RNA m6A in six types of digestive tract tumors. The Kaplan-Meier method and receiver operating characteristic curve were used to evaluate the prognostic and diagnostic value of the newly discovered m6A methyltransferase, ZCCHC4, in CRC. The effects on CRC growth in vitro and in vivo were studied using gain- and loss-of-function experiments. The epigenetic mechanisms underlying ZCCHC4 upregulation in CRC were studied using RIP, MeRIP-seq, RNA pull-down, and animal experiments. RESULTS: We reported that the ZCCHC4-LncRNAGHRLOS-KDM5D axis regulates the growth of CRC in vitro and in vivo. We found that ZCCHC4 was upregulated in primary CRC samples and could predict adverse clinical outcomes in patients with CRC. Mechanistically, ZCCHC4 downregulated LncRNAGHRLOS to promote CRC tumorigenesis. As a downstream molecule of LncRNAGHRLOS, KDM5D directly controls CRC cell proliferation, migration, and invasion. CONCLUSION: This study suggests that the ZCCHC4 axis contributes to the tumorigenesis and progression of CRC and that ZCCHC4 may be a potential biomarker for this malignancy.

CDC123 promotes Hepatocellular Carcinoma malignant progression by regulating CDKAL1.

The cell proliferation protein 123 (CDC123) is involved in the synthesis of the eukaryotic initiation factor 2 (eIF2), which regulates eukaryotic translation. Although CDC123 is considered a candidate oncogene in breast cancer, its expression and role in Hepatocellular Carcinoma (HCC) remain unknown. Herein, we obtained the CDC123 RNA-seq and clinical prognostic data from the TCGA database. The mRNA level revealed that CDC123 was highly expressed in HCC patients, and Kaplan-Meier analysis implied better prognoses in HCC patients with low CDC123 expression (P < 0.001). The multivariate Cox analysis revealed that the CDC123 level was an independent prognostic factor (P < 0.001). We further confirmed a high CDC123 expression in HCC cell lines. Additionally, we found that CDC123 knockdown in HCC cell lines significantly inhibited cellular proliferation, invasion, and migration. Moreover, CDC123 was co-expressed with the CDK5 Regulatory Subunit-Associated Protein 1 Like 1 (CDKAL1), whose mRNA level was decreased after silencing CDC123. Therefore, we hypothesized that CDC123 promotes HCC progression by regulating CDKAL1.

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNATrp, based on the inability of mutants of each enzyme to rescue the growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

NSUN6 Regulates NM23-H1 Expression in an m5C Manner to Affect Epithelial-Mesenchymal Transition in Lung Cancer.

PURPOSE: The expression and regulatory mechanism of NSUN6 in lung cancer are still unclear. Our study explored whether NSUN6 mediates progression of lung cancer by affecting NM23-H1 expression in an m5C-dependent manner. METHODS: qRT-PCR, CCK-8, colony formation, transwell, and Western blot analysis were employed to probe the impact of NSUN6 on lung cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT). RMVar database was utilized to forecast the downstream genes of NSUN6. The mode of interaction between NSUN6 and NM23-H1 was determined by dot blot, luciferase assay, m5C RIP, and cell function assays. The effect of NSUN6 expression on tumor growth was verified in vivo. RESULTS: Expression of NSUN6 was reduced in lung cancer cells, and over-expression of NSUN6 restricted the proliferation of lung cancer cells, migration, and EMT. NSUN6 regulated NM23-H1 expression by modifying the 3'-UTR of NM23-H1 mRNA through m5C and inhibited lung cancer cell proliferation, migration, and EMT. In vivo experiments also showed that over-expression of NSUN6 inhibited the occurrence of lung cancer. CONCLUSION: NSUN6 regulates NM23-H1 expression in an m5C-dependent manner to affect EMT in lung cancer. Thus, NSUN6 may be considered as a potential therapeutic target for lung cancer.

Diversity in Biological Function and Mechanism of the tRNA Methyltransferase Trm10.

ConspectusTransfer ribonucleic acid (tRNA) is the most highly modified RNA species in the cell, and loss of tRNA modifications can lead to growth defects in yeast as well as metabolic, neurological, and mitochondrial disorders in humans. Significant progress has been made toward identifying the enzymes that are responsible for installing diverse modifications in tRNA, revealing a landscape of fascinating biological and mechanistic diversity that remains to be fully explored. Most early discoveries of tRNA modification enzymes were in model systems, where many enzymes were not strictly required for viability, an observation somewhat at odds with the extreme conservation of many of the same enzymes throughout multiple domains of life. Moreover, many tRNA modification enzymes act on more than one type of tRNA substrate, which is not necessarily surprising given the similar overall secondary and tertiary structures of tRNA, yet biochemical characterization has revealed interesting patterns of substrate specificity that can be challenging to rationalize on a molecular level. Questions about how many enzymes efficiently select a precise set of target tRNAs from among a structurally similar pool of molecules persist.The tRNA methyltransferase Trm10 provides an exciting paradigm to study the biological and mechanistic questions surrounding tRNA modifications. Even though the enzyme was originally characterized in Saccharomyces cerevisiae where its deletion causes no detectable phenotype under standard lab conditions, several more recently identified phenotypes provide insight into the requirement for this modification in the overall quality control of the tRNA pool. Studies of Trm10 in yeast also revealed another characteristic feature that has turned out to be a conserved feature of enzymes throughout the Trm10 family tree. We were initially surprised to see that purified S. cerevisiae Trm10 was capable of modifying tRNA substrates that were not detectably modified by the enzyme in vivo in yeast. This pattern has continued to emerge as we and others have studied Trm10 orthologs from Archaea and Eukarya, with enzymes exhibiting in vitro substrate specificities that can differ significantly from in vivo patterns of modification. While this feature complicates efforts to predict substrate specificities of Trm10 enzymes in the absence of appropriate genetic systems, it also provides an exciting opportunity for studying how enzyme activities can be regulated to achieve dynamic patterns of biological tRNA modification, which have been shown to be increasingly important for stress responses and human disease. Finally, the intriguing diversity in target nucleotide modification that has been revealed among Trm10 orthologs is distinctive among known tRNA modifying enzymes and necessitates unusual and likely novel catalytic strategies for methylation that are being revealed by biochemical and structural studies directed toward various family members. These efforts will no doubt yield more surprising discoveries in terms of tRNA modification enzymology.

Functional analysis of tRNA modification enzymes using mutational profiling.

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Function of m5C RNA methyltransferase NOP2 in high-grade serous ovarian cancer.

RNA methyltransferase nucleolar protein p120 (NOP2), commonly referred to as NOP2/Sun RNA methyltransferase family member 1 (NSUN1), is involved in cell proliferation and is highly expressed in various cancers. However, its role in high-grade serous ovarian cancer (HGSOC) remains unclear. Our study investigated the expression of NOP2 in HGSOC tissues and normal fimbria tissues, and found that NOP2 was significantly upregulated in HGSOC tissues. Our experiments showed that NOP2 overexpression promoted cell proliferation in vivo and in vitro and increased the migration and invasion ability of HGSOC cells in vitro. Furthermore, we identified Rap guanine nucleotide exchange factor 4 (RAPGEF4) as a potential downstream target of NOP2 in HGSOC. Finally, our findings suggest that the regulation of NOP2 and RAPGEF4 may depend on m5C methylation levels.

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection.

IMPORTANCE: As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

NOP2-mediated m5C methylation of XPD is associated with hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Our previous study has confirmed that XPD acts as an anti-oncogene and is downregulated in HCC. The mechanism of XPD downregulation in HCC is unclear. In this work, we obtained the datasets related to HCC patients from GSE76427, LIRI-JP, and TCGA-LIHC cohorts. Among 15 m5C regulators (NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, DNMT1, TRDMT1, DNMT3A, DNMT3B and NOP2, TET1, TET2, and TET3, ALYREF), 14 m5C regulators were upregulated in tumor tissues of HCC patients, except for TET2. HCC patients were divided into Cluster A and B with different m5C methylation patterns. Cluster B was enriched in metabolism-related signaling pathways, and Cluster A was prominently associated with the cell cycle signaling pathway. Moreover, XPD was positively correlated with NOP2. Cluster B exhibited upregulation of XPD and had an obvious survival advantage with respect to Cluster A. Additionally, NOP2 and XPD were downregulated in HCC tumors and cells. In vitro assays revealed that NOP2 overexpression enhanced XPD expression by elevating the m5C methylation of XPD, which contributed to inhibit proliferation, migration, and invasion of HCC cells. In conclusion, this work demonstrated that XPD mRNA stability was elevated by NOP2-mediated m5C methylation modification and then inhibited the malignant progression of HCC, suggesting that XPD may be a potential target for HCC treatment.

Biallelic variants in NSUN6 cause an autosomal recessive neurodevelopmental disorder.

PURPOSE: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. METHODS: We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene. RESULTS: We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. CONCLUSION: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.

Human TRMT1 catalyzes m2G or m22G formation on tRNAs in a substrate-dependent manner.

TRMT1 is an N2-methylguanosine (m2G) and N2,N2-methylguanosine (m22G) methyltransferase that targets G26 of both cytoplasmic and mitochondrial tRNAs. In higher eukaryotes, most cytoplasmic tRNAs with G26 carry m22G26, although the majority of mitochondrial G26-containing tRNAs carry m2G26 or G26, suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these tRNAs. Loss-of-function mutations of human TRMT1 result in neurological disorders and completely abrogate tRNA:m22G26 formation. However, the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive, hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1 mutations. Here, we showed that human TRMT1 independently catalyzes formation of the tRNA:m2G26 or m22G26 modification in a substrate-dependent manner, which explains the distinct distribution of m2G26 and m22G26 on cytoplasmic and mitochondrial tRNAs. For human TRMT1-mediated tRNA:m22G26 formation, the semi-conserved C11:G24 serves as the determinant, and the U10:A25 or G10:C25 base pair is also required, while the size of the variable loop has no effect. We defined the requirements of this recognition mechanism as the "m22G26 criteria". We found that the m22G26 modification occurred in almost all the higher eukaryotic tRNAs conforming to these criteria, suggesting the "m22G26 criteria" are applicable to other higher eukaryotic tRNAs.

CDKAL1 Drives the Maintenance of Cancer Stem-Like Cells by Assembling the eIF4F Translation Initiation Complex.

Cancer stem-like cells (CSCs) have a unique translation mode, but little is understood about the process of elongation, especially the contribution of tRNA modifications to the maintenance of CSCs properties. Here, it is reported that, contrary to the initial aim, a tRNA-modifying methylthiotransferase CDKAL1 promotes CSC-factor SALL2 synthesis by assembling the eIF4F translation initiation complex. CDKAL1 expression is upregulated in patients with worse prognoses and is essential for maintaining CSCs in rhabdomyosarcoma (RMS) and common cancers. Translatome analysis reveals that a group of mRNAs whose translation is CDKAL1-dependent contains cytosine-rich sequences in the 5' untranslated region (5'UTR). Mechanistically, CDKAL1 promotes the translation of such mRNAs by organizing the eIF4F translation initiation complex. This complex formation does not require the enzyme activity of CDKAL1 but requires only the NH2 -terminus domain of CDKAL1. Furthermore, sites in CDKAL1 essential for forming the eIF4F complex are identified and discovered candidate inhibitors of CDKAL1-dependent translation.