Nucleotide resolution profiling of m7G tRNA modification by TRAC-Seq

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Abstract

Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m7G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m7G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH4) reduction of m7G sites and subsequent aniline-mediated cleavage of the RNA chain at the resulting abasic sites. The cleaved sites are subsequently ligated with adaptors for the construction of libraries for high-throughput sequencing. The m7G modification sites are identified using a bioinformatic pipeline that calculates the cleavage scores at individual sites on all tRNAs. Unlike antibody-based methods, such as methylated RNA immunoprecipitation and sequencing (meRIP-Seq) for enrichment of methylated RNA sequences, chemically based approaches, including TRAC-Seq, can provide nucleotide-level resolution of modification sites. Compared to the related method AlkAniline-Seq (alkaline hydrolysis and aniline cleavage sequencing), TRAC-Seq incorporates small RNA selection, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution profiling of m7G sites in tRNAs. The m7G TRAC-Seq protocol could be adapted to chemical cleavage–mediated detection of other RNA modifications. The protocol can be completed within ~9 d for four biological replicates of input and treated samples.

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Fig. 1: Partial m7G TRAC-Seq workflow.
Fig. 2: Flowchart summarizing the basic bioinformatic analyses.
Fig. 3: Isolation of small RNAs and total RNA from R1/E mESCs.
Fig. 4: SDS–PAGE analysis of purified recombinant AlkB WT and AlkB D135S.
Fig. 5: Cleavage of an m7G site in tRNA.
Fig. 6: Size distribution of the TRAC-Seq library.
Fig. 7: Anticipated result of TRAC-Seq.

Data availability

The TRAC-Seq data were deposited into the Gene Expression Omnibus database (GEO accession no. GSE112670).

Code availability

The TRAC-seq data analysis source code is available via GitHub (https://github.com/rnabioinfor/TRAC-Seq, https://doi.org/10.5281/zenodo.2671795) and is for research purposes only.

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Acknowledgements

S.L. was supported by grants from the National Natural Science Foundation of China (81772999), the Guangzhou People’s Livelihood Science and Technology Project (201903010006), and a Young Investigator grant from the Alex’s Lemonade Stand Foundation (GR-000000296). R.I.G. was supported by grants from the US National Institute of General Medical Sciences (R01GM086386) and the National Institute of Mental Health (R21MH118594).

Author information

S.L. developed the protocol. S.L. and Y.-Z.J. performed the experiments. Q.L. designed the bioinformatic pipeline and analyzed the data. S.L., Q.L., and R.I.G. wrote the manuscript.

Correspondence to Shuibin Lin or Richard I. Gregory.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Frank Lyko and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Lin, S. et al. Mol. Cell 71, 244–255.e5 (2018) https://doi.org/10.1016/j.molcel.2018.06.001

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Lin, S., Liu, Q., Jiang, Y. et al. Nucleotide resolution profiling of m7G tRNA modification by TRAC-Seq. Nat Protoc 14, 3220–3242 (2019) doi:10.1038/s41596-019-0226-7

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