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Oxford University Press
Nucleic Acids Research
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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 (m1A) 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, m1A 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 m1A, 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 m1A in the template RNA, meaning it is sequence dependent. The RT-signature ofm1Awas used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.
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Hauenschild, Ralf; Tserovski, Lyudmil; Schmid, Katharina; Thuring, Kathrin; Winz, Marie-Luise; Sharma, Sunny; Entian, Karl-dieter; Wacheul, Ludvine; Lafontaine, Denis L.J.; Anderson, James T.; Alfonzo, Juan; Hildebrandt, Andreas; Jaschke, Andres; Motorin, Yuri; and Helm, Mark, "The Reverse Transcription Signature of N-1-Methyladenosine in RNA-Seq is Sequence Dependent" (2015). Biological Sciences Faculty Research and Publications. 509.