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

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

Author information

1
Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, USA.
2
Department of Biochemistry &Biophysics, University of Rochester School of Medicine, Rochester, New York, USA.
3
Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA.

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.

PMID:
26237225
PMCID:
PMC4553111
DOI:
10.1038/nmeth.3508
[Indexed for MEDLINE]
Free PMC Article

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