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Background <p>Transfer RNA (tRNA) modifications play important roles in regulating gene expression and protein synthesis, yet their dynamic interplay remains elusive. The human tRNAome contains approximately 40 modification types, distributed on average at 13 sites in cytosolic tRNAs and 6 sites in mitochondrial tRNAs. tRNA modifications exhibit dynamic patterns dependent on cell types and cell states, underscoring the need for advancing methodologies to assess their variations and crosstalk transcriptome-wide.</p> Results <p>Here, we develop enhanced single-read analysis of tRNA crosstalks (eSLAC), an integrative platform combining multiplex small RNA sequencing (MSR-seq) with expanded detection of pseudouridine (Ψ), 5-formylcytidine (f<sup>5</sup>C), N4-acetylcytidine (ac<sup>4</sup>C), and a single-read analysis pipeline to map modifications and their crosstalk (eSLAC). eSLAC enables the detection of over 60% of all human tRNA modification sites and assigns sites to three Ψ writer enzymes. The analysis reveals strong positive Ψ-Ψ and Ψ-charging crosstalks. Applying polysome tRNA profiling, we identify differential tRNA isodecoder usage and site-specific tRNA Ψ variations on the polysome.</p> Conclusions <p>This study establishes a framework for dissecting the interconnected architecture of tRNA modome and its functional complexity.</p>

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Decoding human tRNA modifications and crosstalk by enhanced single-read analysis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月20日

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  • Mahdi Assari,
  • Brandon J. Chew,
  • Mohammad Amin Bayat Tork,
  • Marek Sobczyk,
  • Chenyou Zhu,
  • Hoang Anh V. Tran,
  • Dominika Rudzka,
  • Wen Zhang,
  • Tao Pan

摘要

Background

Transfer RNA (tRNA) modifications play important roles in regulating gene expression and protein synthesis, yet their dynamic interplay remains elusive. The human tRNAome contains approximately 40 modification types, distributed on average at 13 sites in cytosolic tRNAs and 6 sites in mitochondrial tRNAs. tRNA modifications exhibit dynamic patterns dependent on cell types and cell states, underscoring the need for advancing methodologies to assess their variations and crosstalk transcriptome-wide.

Results

Here, we develop enhanced single-read analysis of tRNA crosstalks (eSLAC), an integrative platform combining multiplex small RNA sequencing (MSR-seq) with expanded detection of pseudouridine (Ψ), 5-formylcytidine (f5C), N4-acetylcytidine (ac4C), and a single-read analysis pipeline to map modifications and their crosstalk (eSLAC). eSLAC enables the detection of over 60% of all human tRNA modification sites and assigns sites to three Ψ writer enzymes. The analysis reveals strong positive Ψ-Ψ and Ψ-charging crosstalks. Applying polysome tRNA profiling, we identify differential tRNA isodecoder usage and site-specific tRNA Ψ variations on the polysome.

Conclusions

This study establishes a framework for dissecting the interconnected architecture of tRNA modome and its functional complexity.