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Cell-type-specific expression of tRNAs in the brain regulates cellular homeostasis

ABSTRACT: Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA Polymerase III is conditionally epitope-tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in eleven diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA anticodon pools is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA anticodon families. Cell type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.

ORGANISM(S): Mus musculus

PROVIDER: GSE239759 | GEO | 2024/02/19

REPOSITORIES: GEO

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Cell type-specific expression of tRNAs in the brain regulates cellular homeostasis.

Project description:Defects in tRNA biogenesis are associated with diverse neurological disorders, yet our understanding of these diseases has been hampered an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA Polymerase III is conditionally epitope-tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in eleven diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA anticodon pools is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA anticodon families. Cell type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.

2024-02-19 | GSE250090 | GEO

Small RNA sequencing of in vitro Angiogenin-hydrolyzed tRNAs

Project description:Specific environmental insults cause the limited fragmentation of transfer RNAs (tRNAs) into tRNA-derived small RNAs (tsRNAs), which have been implicated in a wide range of biological processes. tRNA fragmentation results from endonucleolytic activities targeting single-stranded tRNA regions. However, how a tRNA with a single hydrolyzed phosphodiester bond in the anticodon loop (‘nicked’ tRNA) gives rise to distinct tsRNAs remains poorly understood. After identifying RNA helicases that were able to unwind ‘nicked’ tRNAs in vitro, the specificity of one of those enzymes, DDX3X, was determined by RNA helicase assays on tRNAs, which had been subjected to recombinant Angiogenin. Both in vivo ‘nicked’ tRNAs, DDX3X-unwound ‘nicked’ tRNAs as well as heat-denatured ‘nicked’ tRNAs were subjected to small RNA sequencing.

2022-04-05 | GSE187021 | GEO

Identification of RNA helicases that mediate processing of tRNAs into distinct tRNA fragments by LC-MS/MS.

Project description:Specific environmental insults cause the limited fragmentation of transfer RNAs (tRNAs) into tRNA-derived small RNAs (tsRNAs), which have been implicated in a wide range of biological processes. tRNA fragmentation results from endonucleolytic activities targeting single-stranded tRNA regions. However, how a tRNA with a single hydrolyzed phosphodiester bond in the anticodon loop (‘nicked’ tRNA) gives rise to distinct tsRNAs remains poorly understood. By utilizing biochemical fractionation of tsRNA-containing ribonucleoprotein complexes (RNPs) coupled with LC-MS/MS, we identified several RNA helicase enzymes that represent putative tRNA/tsRNA processing enzymes. Furthermore, a combination of biochemical and computational approaches revealed that specific RNA helicases indeed bind specific tRNAs with high affinity, as well as process nicked tRNAs into individual tsRNAs. In summary, these findings reveal that tRNA-derived duplexes can be substrates of well-known RNA helicases, thereby expanding their potential for cellular function to the creation of individual tsRNAs during the cellular stress response.

2023-01-09 | PXD029869 | Pride

The modification landscape of P. aeruginosa tRNAs

Project description:RNA modifications have a substantial impact on tRNA function. While modifications in the anticodon loop play an important role in translational fidelity, modifications in the tRNA core influence tRNA structural stability. In bacteria, tRNA modifications play important roles in the stress response and expression of virulence factors. While tRNA modifications are well characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa is lacking. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which we used to identify tRNA modifications in P. aeruginosa. We determined conservation for many modifications between the two organisms. We also identified potential sites of tRNA modification in P. aeruginosa tRNA that are not present in E. coli. One of these sites is found at the same positions as acacp3U, a modification previously identified in Vibrio cholerae. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which may play roles in determining virulence and pathogenicity.

2024-04-03 | GSE236676 | GEO

Queuosine modification protects cognate tRNAs against ribonuclease cleavage

Project description:Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multi-cellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pool in human cells.

2018-08-10 | GSE102570 | GEO

Dynamic modification of tRNAs determines pathogenicity in the opportunistic pathogen Pseudomonas aeruginosa [RNA-Seq and Ribo-Seq]

Project description:The overall success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon alter ribosome occupancy of transcripts enriched in cognate codons of such tRNAs. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications influenced expression of genes encoding virulence regulators, leading to a cellular proteomic shift towards pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to changing and challenging habitats prevailing in the host niche.

2024-02-18 | GSE233297 | GEO

RNA-Seq analysis for the study "Dynamic modification of tRNAs determines pathogenicity in the opportunistic pathogen Pseudomonas aeruginosa"

Project description:The overall success of a pathogenic microbe depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered and often not completely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Growth phase-dependent enzymatic activity of GidA leads to differential introduction of carboxymethylaminomethyl-uridine (cmnm5U34) at uridine 34 of the anticodon of selected tRNAs. Consequently, ribosome occupancy of transcripts enriched in cognate codons of such tRNAs is enhanced. Specifically, the presence of GidA-dependent tRNA modifications influenced expression of genes encoding for virulence factors leading to a cellular proteomic shift towards pathogenic and well adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept and provides a paradigm of how tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to changing and challenging habitats prevailing in the host niche.

2024-02-18 | GSE149304 | GEO

METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

Project description:A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear-encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.

2022-05-19 | PXD030418 | Pride

tRNAs marked with CCACCA are targeted for degradation

Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included

2011-11-17 | E-GEOD-33756 | biostudies-arrayexpress

Transfer RNA pools in human cells are controlled by selective gene expression [tRNA-Seq]

Project description:Transfer RNAs are required for translating genetic information into protein sequence. The human genome contains hundreds of tRNA genes, many of which in multiple copies. How their expression is regulated to control functional tRNA levels is unknown. Here, we combined quantitative tRNA profiling and ChIP-Seq to measure tRNA expression upon differentiation of human induced pluripotent stem cells (hiPSC) into neuronal and cardiac cells. We find that tRNA transcript pools vary substantially, while the abundance of tRNAs with distinct anticodons, which governs decoding rates, is more stable among cell types. Mechanistically, RNA Polymerase III (Pol III) samples a wide range of tRNA genes in hiPSC and becomes constrained to a housekeeping subset upon differentiation. This is mediated by diminished mTOR signaling, which activates the Pol III repressor MAF1. Our data rationalize how tRNA anticodon pools are buffered in different cellular contexts and reveal that mTOR activity drives selective tRNA expression.

2023-10-23 | GSE227927 | GEO

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