Transcriptome‐Wide Identification of Pseudouridine Modifications Using Pseudo‐seq

A diverse array of post‐transcriptional modifications is found in RNA molecules from all domains of life. While the locations of RNA modifications are well characterized in abundant noncoding RNAs, modified sites in less abundant mRNAs are just beginning to be discovered. Recent work has revealed hundreds of previously unknown and dynamically regulated pseudouridines (Ψ) in mRNAs from diverse organisms. This unit describes Pseudo‐seq, an efficient, high‐resolution method for identification of Ψs genome‐wide. This unit includes methods for isolation of RNA from S. cerevisiae, preparation of Pseudo‐seq libraries from RNA samples, and identification of sites of pseudouridylation from the sequencing data. Pseudo‐seq is applicable to any organism or cell type, facilitating rapid identification of novel pseudouridylation events. © 2015 by John Wiley & Sons, Inc.

[1]  N. Ho,et al.  Reaction of pseudouridine and inosine with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide. , 1971, Biochemistry.

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

[3]  M. W. Gray,et al.  Pseudouridine in RNA: What, Where, How, and Why , 2000, IUBMB life.

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

[5]  F. Davis,et al.  Ribonucleic acids from yeast which contain a fifth nucleotide. , 1957, The Journal of biological chemistry.

[6]  O. Elemento,et al.  Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons , 2012, Cell.

[7]  D. Bartel,et al.  Poly(A)-tail profiling reveals an embryonic switch in translational control , 2014, Nature.

[8]  R. Gupta,et al.  Rapid print-readout technique for sequencing of RNA's containing modified nucleotides. , 1979, Nucleic acids research.

[9]  G. Church,et al.  Genome-Wide Identification of Human RNA Editing Sites by Parallel DNA Capturing and Sequencing , 2009, Science.

[10]  John Karijolich,et al.  Modifying the genetic code: Converting nonsense codons into sense codons by targeted pseudouridylation , 2011, Nature.

[11]  J Ofengand,et al.  Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. , 1993, Biochemistry.

[12]  Yi-Tao Yu,et al.  U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP , 2011, The EMBO journal.

[13]  Niki S. C. Wong,et al.  28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures. , 2014, Journal of biotechnology.

[14]  T. Tuschl,et al.  Cloning of Small RNA Molecules , 2003, Current protocols in molecular biology.

[15]  J. T. Madison,et al.  NUCLEOTIDE SEQUENCES IN THE YEAST ALANINE TRANSFER RIBONUCLEIC ACID. , 1965, The Journal of biological chemistry.

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

[17]  Yi-Tao Yu,et al.  RNA pseudouridylation: new insights into an old modification. , 2013, Trends in biochemical sciences.

[18]  Jef Rozenski,et al.  The RNA modification database, RNAMDB: 2011 update , 2010, Nucleic Acids Res..

[19]  Joshua A. Arribere,et al.  Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing , 2013, Genome research.

[20]  M. Collart,et al.  Preparation of Yeast RNA , 1993, Current protocols in molecular biology.

[21]  P. Brown,et al.  Transcriptome-Wide Mapping of Pseudouridines: Pseudouridine Synthases Modify Specific mRNAs in S. cerevisiae , 2014, PloS one.

[22]  Yi-Tao Yu,et al.  U 2 snRNA is inducibly pseudouridylated at novel sites by Pus 7 p and snR 81 RNP , 2010 .

[23]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[24]  L. Steinmetz,et al.  Bidirectional promoters generate pervasive transcription in yeast , 2009, Nature.

[25]  Graham A. Hudson,et al.  Thermodynamic contribution and nearest-neighbor parameters of pseudouridine-adenosine base pairs in oligoribonucleotides , 2013, RNA.

[26]  V. Ramakrishnan,et al.  Unusual base pairing during the decoding of a stop codon by the ribosome , 2013, Nature.

[27]  H. Busch,et al.  Modified nucleotides in T1 RNase oligonucleotides of 18S ribosomal RNA of the Novikoff hepatoma. , 1978, Biochemistry.

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

[29]  W. Cohn.  Pseudouridine, a carbon-carbon linked ribonucleoside in ribonucleic acids: isolation, structure, and chemical characteristics. , 1960, The Journal of biological chemistry.