ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

Nat Methods. 2015 Sep;12(9):879-84. doi: 10.1038/nmeth.3508. Epub 2015 Aug 3.

Abstract

High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Algorithms*
  • Base Sequence
  • DNA Methylation / genetics*
  • Escherichia coli Proteins / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Mixed Function Oxygenases / genetics*
  • Molecular Sequence Data
  • RNA, Transfer / genetics*
  • Software*

Substances

  • Escherichia coli Proteins
  • RNA, Transfer
  • Mixed Function Oxygenases
  • AlkB protein, E coli