Efficient and quantitative high-throughput transfer RNA sequencing

Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.

[1]  Y. Mishina,et al.  Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli. , 2004, Journal of the American Chemical Society.

[2]  S. Levine,et al.  Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number , 2014, Nucleic acids research.

[3]  Sebastian M. Waszak,et al.  A Dual Program for Translation Regulation in Cellular Proliferation and Differentiation , 2014, Cell.

[4]  Ye Fu,et al.  Nucleic Acid Oxidation in DNA Damage Repair and Epigenetics , 2014, Chemical reviews.

[5]  T. Pan,et al.  tRNA over-expression in breast cancer and functional consequences , 2009, Nucleic acids research.

[6]  Z. Ignatova,et al.  Emerging roles of tRNA in adaptive translation, signalling dynamics and disease , 2014, Nature Reviews Genetics.

[7]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[8]  T. Hollis,et al.  Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching , 2010, PloS one.

[9]  V. Iyer,et al.  Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing , 2013, RNA.

[10]  C. Francklyn,et al.  Transfer RNA and human disease , 2014, Front. Genet..

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

[12]  Tao Pan,et al.  Tissue-Specific Differences in Human Transfer RNA Expression , 2006, PLoS genetics.

[13]  Arne Klungland,et al.  ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. , 2013, Molecular cell.

[14]  Adam Frost,et al.  Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains , 2015, Science.

[15]  Sue Povey,et al.  Gene map of the extended human MHC , 2004, Nature Reviews Genetics.

[16]  P. Weber,et al.  Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB , 2006, Nature.

[17]  Erling Seeberg,et al.  AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli , 2002, Nature.

[18]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[19]  Yidan Qin,et al.  Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5 , 2014, Proceedings of the National Academy of Sciences.

[20]  T. Pan,et al.  Diversity of tRNA genes in eukaryotes , 2006, Nucleic acids research.

[21]  Robert P. Hausinger,et al.  Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage , 2002, Nature.

[22]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .