2. Academic Validation
  3. Nucleotide resolution profiling of m3C RNA modification by HAC-seq

Nucleotide resolution profiling of m3C RNA modification by HAC-seq

  • Nucleic Acids Res. 2021 Mar 18;49(5):e27. doi: 10.1093/nar/gkaa1186.
Jia Cui 1 2,  Qi Liu 1 2,  Erdem Sendinc 3,  Yang Shi 3 4,  Richard I Gregory 1 2 5 6 7
Affiliations

Affiliations

  • 1 Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.
  • 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
  • 3 Division of Newborn Medicine and Epigenetics Program, Department of Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
  • 4 Ludwig Institute for Cancer Research, Oxford Branch, Oxford University, Oxford OX3 7DQ, UK.
  • 5 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
  • 6 Harvard Initiative for RNA Medicine, Boston, MA 02115, USA.
  • 7 Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
PMID: 33313824 DOI: 10.1093/nar/gkaa1186
Abstract

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called 'epitranscriptome', contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage Sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

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