Publication Date:
2015-11-17
Description:
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.
Keywords:
Nucleic acid modification
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
