Functional analysis of tRNA modification enzymes using mutational profiling

Yamagami, Ryota; Hori, Hiroyuki

doi:10.1016/bs.mie.2023.02.021

Cited by 2 publications

(4 citation statements)

References 58 publications

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“…For the high-throughput analysis of MTR1, we prepared an oPools (Integrated DNA Technologies) DNA oligonucleotide mixture of 141 distinct MTR1 variants that have a single point nucleotide substitution in the core domain (Supplementary Material S1) as described previously (Yamagami and Hori, 2023b). The MTR1 reaction was performed in a 50 µL mixture containing TAM buffer (pH 6.5), 5 mM MgCl 2 , 100 µM m 6 G, 120 mM KCl, and 8.5 µM MTR1 mixture at 37 °C for 3 h. The RNA mixture was purified by phenol extraction and recovered by ethanol precipitation.…”

Section: Detection Of the M 1 A Formation By Mutational Profilingmentioning

confidence: 99%

“…As a negative control experiment, the reaction without m 6 G was performed. The RNA was dephosphorylated by treatment with rSAP (New England Biolabs) and libraries for next generation sequencing were prepared by reverse transcription with TGIRT (InGex) at 42 °C for 16 h, circligation, and indexing PCR as described previously (Yamagami and Hori, 2023b). Single-end sequencing of the DNA library was done on a NextSeq 550 (Illumina) using 150 cycles, mid-output flow cells, and the standard Illumina sequencing primer.…”

Section: Detection Of the M 1 A Formation By Mutational Profilingmentioning

confidence: 99%

“…Mutational profiling at A11 in each MTR1 variant was conducted as described previously (Yamagami and Hori, 2023a;Yamagami and Hori, 2023b). In brief, the 3′ adapter sequence was removed from sequence reads using Cutadapt (Martin, 2011) with the following command: cutadapt -a CTGTAGGCACCATCAAT--nextseq-trim = 30 -j 0 -o output.fastq.gz input.fastq.gz.…”

Section: Mutational Profilingmentioning

confidence: 99%

“…These mutations in the cDNA can be detected by next-generation sequencing (Yamagami et al, 2022), providing positional and quantitative information on methylated nucleosides in RNA. Very recently, we developed a mutational profiling technique that was linked to high-throughput functional analysis of tRNA methyltransferase, called tRNA-MaP (Yamagami and Hori, 2023a;Yamagami and Hori, 2023b).…”

Section: Introductionmentioning

confidence: 99%

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High-throughput mutational analysis of a methyltransferase ribozyme

Yamagami,

Kubota,

Kohno

et al. 2024

Front. RNA Res.

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Methyltransferase ribozyme 1 (MTR1) is a catalytic RNA that has been isolated from a random RNA pool by in vitro selection. The ribozyme catalyzes site-specific formation of 1-methyl adenosine (m1A) using 6-methyl guanine (m6G) as a methyl group donor. The ribozyme has been extensively characterized by biochemical and structural analyses. Here, we describe high-throughput screening of single point mutants in the catalytic domain of MTR1 and determine their effect on ribozyme activity. Our mutational profiling method successfully assessed the activity of the 141 MTR1 variants tested in each experiment and revealed that the ribozyme is very sensitive to nucleotide substitutions in the catalytic core domain. Our technique can be applied to methyltransferase ribozymes that catalyze formation of different modifications such as 7-methylguanosine (m7G) or 3-methylcytidine (m3C).

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Section: Detection Of the M 1 A Formation By Mutational Profilingmentioning

confidence: 99%

Section: Detection Of the M 1 A Formation By Mutational Profilingmentioning

confidence: 99%

Section: Mutational Profilingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

High-throughput mutational analysis of a methyltransferase ribozyme

Yamagami,

Kubota,

Kohno

et al. 2024

Front. RNA Res.

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tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators

Li,

Yu,

Jiang

et al. 2024

Biomolecules

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As the most ancient RNA, transfer RNAs (tRNAs) play a more complex role than their constitutive function as amino acid transporters in the protein synthesis process. The transcription and maturation of tRNA in cells are subject to stringent regulation, resulting in the formation of tissue- and cell-specific tRNA pools with variations in tRNA overall abundance, composition, modification, and charging levels. The heterogeneity of tRNA pools contributes to facilitating the formation of histocyte-specific protein expression patterns and is involved in diverse biological processes. Moreover, tRNAs can be recognized by various RNase under physiological and pathological conditions to generate tRNA-derived small RNAs (tsRNAs) and serve as small regulatory RNAs in various biological processes. Here, we summarize these recent insights into the heterogeneity of tRNA and highlight the advances in the regulation of tRNA function and tsRNA biogenesis by tRNA modifications. We synthesize diverse mechanisms of tRNA and tsRNA in embryonic development, cell fate determination, and epigenetic inheritance regulation. We also discuss the potential clinical applications based on the new knowledge of tRNA and tsRNA as diagnostic and prognostic biomarkers and new therapeutic strategies for multiple diseases.

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Functional analysis of tRNA modification enzymes using mutational profiling

Cited by 2 publications

References 58 publications

High-throughput mutational analysis of a methyltransferase ribozyme

High-throughput mutational analysis of a methyltransferase ribozyme

tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators

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