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

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.

[1]  W. Gilbert,et al.  Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells , 2014, Nature.

[2]  P. Falnes.  Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins. , 2004, Nucleic acids research.

[3]  E. Phizicky,et al.  Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding , 2009, Journal of Structural and Functional Genomics.

[4]  J. Yong,et al.  tRNA binds to cytochrome c and inhibits caspase activation. , 2010, Molecular cell.

[5]  Steve Hoffmann,et al.  Traces of post-transcriptional RNA modifications in deep sequencing data , 2011, Biological chemistry.

[6]  Tsutomu Suzuki,et al.  A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs , 2014, Nucleic acids research.

[7]  angesichts der Corona-Pandemie,et al.  UPDATE , 1973, The Lancet.

[8]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[9]  Huihao Zhou,et al.  Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration , 2014, Science.

[10]  Yuk Yee Leung,et al.  HAMR: high-throughput annotation of modified ribonucleotides , 2013, RNA.

[11]  Markus Glatzel,et al.  CLP1 links tRNA metabolism to progressive motor-neuron loss , 2013, Nature.

[12]  E. Phizicky,et al.  A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop. , 2011, RNA.

[13]  Marcin Feder,et al.  MODOMICS: a database of RNA modification pathways , 2005, Nucleic Acids Res..

[14]  Francesca Tuorto,et al.  RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. , 2010, Genes & development.

[15]  Hsien-Da Huang,et al.  RNAcentral: an international database of ncRNA sequences , 2014, Nucleic Acids Res..

[16]  A. Malhotra,et al.  A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). , 2009, Genes & development.

[17]  A. Hopper,et al.  tRNA biology charges to the front. , 2010, Genes & development.

[18]  Patricia P. Chan,et al.  GtRNAdb: a database of transfer RNA genes detected in genomic sequence , 2008, Nucleic Acids Res..

[19]  T. Speed,et al.  A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus. , 2012, Molecular cell.

[20]  G. Barton,et al.  Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. , 2009, RNA.

[21]  A. Hinnebusch,et al.  The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA. , 1998, Genes & development.

[22]  Andrea Califano,et al.  tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma , 2013, Proceedings of the National Academy of Sciences.

[23]  J. Bujnicki,et al.  MODOMICS: a database of RNA modification pathways—2013 update , 2012, Nucleic Acids Res..

[24]  Praveen Sethupathy,et al.  Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C , 2015, Scientific Reports.

[25]  E. D. De Robertis,et al.  RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. , 1981, Journal of molecular biology.

[26]  R. Parker,et al.  Stressing Out over tRNA Cleavage , 2009, Cell.

[27]  T. Preiss,et al.  Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA , 2012, Nucleic acids research.

[28]  A. Sandelin,et al.  Hidden layers of human small RNAs , 2008, BMC Genomics.

[29]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[30]  Herbert H. Tsang,et al.  Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications , 2009, Nucleic acids research.

[31]  Robert W Taylor,et al.  Mitochondrial tRNA mutations and disease , 2010, Wiley interdisciplinary reviews. RNA.

[32]  S. Eddy,et al.  tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. , 1997, Nucleic acids research.

[33]  D. Haussecker,et al.  Human tRNA-derived small RNAs in the global regulation of RNA silencing. , 2010, RNA.

[34]  A. Hopper.  Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae , 2013, Genetics.

[35]  M. Tuite,et al.  tRNA and protein methylase complexes mediate zymocin toxicity in yeast , 2008, Molecular microbiology.

[36]  J. Essigmann,et al.  Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Magnar Bjørås,et al.  Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA , 2003, Nature.

[38]  Jernej Ule,et al.  Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders , 2014, The EMBO journal.

[39]  Maxwell R. Mumbach,et al.  Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA , 2014, Cell.

[40]  Frank Lyko,et al.  RNA cytosine methylation analysis by bisulfite sequencing , 2008, Nucleic acids research.

[41]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[42]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[43]  Christiane Branlant,et al.  Identification of modified residues in RNAs by reverse transcription-based methods. , 2007, Methods in enzymology.