Portal de Periódicos da CAPES

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

2015; Nature Portfolio; Volume: 12; Issue: 9 Linguagem: Inglês

10.1038/nmeth.3508

1548-7105

Aaron E. Cozen, Erin Quartley, Andrew Holmes, Eva Hrabeta‐Robinson, Eric M. Phizicky, Todd M. Lowe,

Cancer-related molecular mechanisms research

ARM-seq enables enhanced sequencing of modified tRNAs and tRNA fragments. Treatment of RNA with the demethylase AlkB prior to reverse transcription removes three ‘hard-stop’ modifications, allowing for discovery of modified tRNA fragments and their precursors by RNA sequencing. 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 N1-methyladenosine (m1A), N3-methylcytidine (m3C) and N1-methylguanosine (m1G), 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 m1A 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.