2. Academic Validation
  3. The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent

The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent

  • Nucleic Acids Res. 2015 Nov 16;43(20):9950-64. doi: 10.1093/nar/gkv895.
Ralf Hauenschild 1,  Lyudmil Tserovski 1,  Katharina Schmid 1,  Kathrin Thüring 1,  Marie-Luise Winz 2,  Sunny Sharma 3,  Karl-Dieter Entian 3,  Ludivine Wacheul 4,  Denis L J Lafontaine 4,  James Anderson 5,  Juan Alfonzo 6,  Andreas Hildebrandt 7,  Andres Jäschke 2,  Yuri Motorin 8,  Mark Helm 9
Affiliations

Affiliations

  • 1 Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
  • 2 Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
  • 3 Institute of Molecular Biosciences: Goethe University Frankfurt, Max-von-Laue Street 9, 60438 Frankfurt/M, Germany.
  • 4 RNA Molecular Biology, Université Libre de Bruxelles, Rue Profs Jeener & Brachet, 12, B-6041 Charleroi-Gosselies, Belgium.
  • 5 Department of Biological Sciences, Marquette University, 53201-1881, Milwaukee, WI, USA.
  • 6 Department of Microbiology, The Ohio State University, 43210, Columbus, OH, USA.
  • 7 Institute for Computer Sciences, Johannes Gutenberg University Mainz, Staudingerweg 9, 55128 Mainz, Germany.
  • 8 IMoPA UMR7365 CNRS-UL, BioPole de l'Université de Lorraine, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France motorine5@univ-lorraine.fr.
  • 9 Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany mhelm@uni-mainz.de.
PMID: 26365242 DOI: 10.1093/nar/gkv895
Abstract

The combination of Reverse Transcription (RT) and high-throughput Sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m(1)A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of Antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m(1)A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m(1)A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3' of m(1)A in the template RNA, meaning it is sequence dependent. The RT-signature of m(1)A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m(1)A residues in trypanosomal tRNA.

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