title: The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent.
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.
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hauenschild
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Lyudmil
authors.initials: L
authors.lastname: Tserovski
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schmid
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Thüring
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Winz
authors.affiliation: Institute of Molecular Biosciences: Goethe University Frankfurt, Max-von-Laue Street 9, 60438 Frankfurt/M, Germany.
authors.forename: Sunny
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Institute of Molecular Biosciences: Goethe University Frankfurt, Max-von-Laue Street 9, 60438 Frankfurt/M, Germany.
authors.forename: Karl-Dieter
authors.initials: KD
authors.lastname: Entian
authors.affiliation: RNA Molecular Biology, Université Libre de Bruxelles, Rue Profs Jeener & Brachet, 12, B-6041 Charleroi-Gosselies, Belgium.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Wacheul
authors.affiliation: RNA Molecular Biology, Université Libre de Bruxelles, Rue Profs Jeener & Brachet, 12, B-6041 Charleroi-Gosselies, Belgium.
authors.forename: Denis L J
authors.initials: DL
authors.lastname: Lafontaine
authors.affiliation: Department of Biological Sciences, Marquette University, 53201-1881, Milwaukee, WI, USA.
authors.forename: James
authors.initials: J
authors.lastname: Anderson
authors.affiliation: Department of Microbiology, The Ohio State University, 43210, Columbus, OH, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Alfonzo
authors.affiliation: Institute for Computer Sciences, Johannes Gutenberg University Mainz, Staudingerweg 9, 55128 Mainz, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hildebrandt
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
authors.affiliation: 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.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany mhelm@uni-mainz.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000069550:Machine Learning; D051379:Mice; D012313:RNA / Q000737:chemistry*; D048348:Reverse Transcription; D017423:Sequence Analysis, RNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: C002230:1-methyladenosine; D012313:RNA; D000241:Adenosine