RESUMO
Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.
Assuntos
Ceco/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiota/genética , RNA de Transferência/genética , Análise de Sequência de RNA/métodos , Animais , Bacillus subtilis/genética , Bacteroidetes/genética , Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/genéticaRESUMO
The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Pseudouridina/química , Pseudouridina/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/química , RNA Ribossômico/genética , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , CME-Carbodi-Imida/análogos & derivados , DNA Complementar/genética , Células HEK293 , Humanos , Mutação , Pseudouridina/metabolismo , DNA Polimerase Dirigida por RNA/química , Transcrição Reversa , Alinhamento de SequênciaRESUMO
Fidelity of translation, which is predominately dictated by the accuracy of aminoacyl-tRNA synthetases in pairing amino acids with correct tRNAs, is of central importance in biology. Yet, deliberate modifications of translational fidelity can be beneficial. Here we found human and not E. coli AlaRS has an intrinsic capacity for mispairing alanine onto nonalanyl-tRNAs including tRNACys. Consistently, a cysteine-to-alanine substitution was found in a reporter protein expressed in human cells. All human AlaRS-mischarged tRNAs have a G4:U69 base pair in the acceptor stem. The base pair is required for the mischarging. By solving the crystal structure of human AlaRS and comparing it to that of E. coli AlaRS, we identified a key sequence divergence between eukaryotes and bacteria that influences mischarging. Thus, the expanded tRNA specificity of AlaRS appears to be an evolutionary gain-of-function to provide posttranscriptional alanine substitutions in eukaryotic proteins for potential regulations.
