Mutations in the tRNA methyltransferase 1 (TRMT1) gene have been identified as the cause of certain forms of autosomal-recessive intellectual disability (ID). However, the molecular pathology ...underlying ID-associated TRMT1 mutations is unknown, since the biological role of the encoded TRMT1 protein remains to be determined. Here, we have elucidated the molecular targets and function of TRMT1 to uncover the cellular effects of ID-causing TRMT1 mutations. Using human cells that have been rendered deficient in TRMT1, we show that TRMT1 is responsible for catalyzing the dimethylguanosine (m2,2G) base modification in both nucleus- and mitochondrion-encoded tRNAs. TRMT1-deficient cells exhibit decreased proliferation rates, alterations in global protein synthesis, and perturbations in redox homeostasis, including increased endogenous ROS levels and hypersensitivity to oxidizing agents. Notably, ID-causing TRMT1 variants are unable to catalyze the formation of m2,2G due to defects in RNA binding and cannot rescue oxidative stress sensitivity. Our results uncover a biological role for TRMT1-catalyzed tRNA modification in redox metabolism and show that individuals with TRMT1-associated ID are likely to have major perturbations in cellular homeostasis due to the lack of m2,2G modifications.
The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA ...methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare ...and do not easily provide information on the temporal nature of events. Here, we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNA
with time-resolved NMR measurements, we show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we show that a strong hierarchy controls the introduction of the T54, Ψ55 and m
A58 modifications in the T-arm, and we demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.
Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the ...investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNA
and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.
The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ₀) synthesis and DNA metabolism ...genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2′-deoxy-preQ₀ and 2′-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ₀ derivative, 2′-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.
The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that ...originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.
Mutations in the cytosine‐5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post‐transcriptional methylation contributes to the human ...disease is currently unknown. By comparing gene expression data with global cytosine‐5 RNA methylomes in patient fibroblasts and NSun2‐deficient mice, we find that loss of cytosine‐5 RNA methylation increases the angiogenin‐mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5′ tRNA‐derived small RNA fragments. Accumulation of 5′ tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site‐specific NSun2‐mediated methylation and that the presence of 5′ tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2‐deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2‐mediated tRNA methylation contributes to human diseases via stress‐induced RNA cleavage.
Synopsis
This study causally links post‐transcriptional methylation‐controlled tRNA identity and their stability to neurological disorders in human.
NSun2‐mediated tRNA methylation protects from endonucleolytic cleavage into small RNA fragments.
tRNA‐derived small RNA fragments are sufficient and required to induce cellular stress responses.
Loss of cytosine‐5 methylation in tRNAs contributes to neuro‐developmental disease through accumulation of tRNA‐derived small RNA fragments.
This study causally links post‐transcriptional methylation‐controlled tRNA identity and their stability to neurological disorders in human.
Nucleic acids, especially RNA, naturally contain a diversity of chemically modified nucleosides. To understand the biological role of these modified nucleosides, nucleic acid scientists need tools to ...specifically label, detect and enrich modified nucleic acids. These tools comprise a diverse set of chemical reagents which have been established in the early years of nucleic acid research. Recent developments in high-throughput sequencing and mass spectrometry utilize these chemical labeling strategies to efficiently detect and localize modifications in nucleic acids. As a consequence the transcriptome-wide distribution of modified nucleosides, especially 5-methylcytosine and pseudouridine, in all domains of life could be analyzed. With the help of these techniques and the gained knowledge, it becomes possible to understand the functions of modifications and even study their connections to human health and disease. Here, the differential chemical reactivity of modified nucleosides and their canonical counterpart is reviewed and discussed.
Modified nucleotide 5-methylcytosine (m5C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m5C-methyltranferases ...(MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m5C- MTase activity, which restores m5C formation at position 2870 in domain V of 25S rRNA in a nop2Δ yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m5C in domains V and IV, respectively. In addition, we do not find any evidence of m5C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m5C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m5C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m5C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the ...wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the
locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.
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