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 (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 of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.
Highlights
Like in conventional RNASeq procedures, RNA preparations were reverse transcribed into cDNA libraries and submitted to Illumina sequencing
Because certain sequence contexts were over-represented in that dataset, we further reduced the dataset by averaging data points from RNAs of over 95% sequence identity and a final averaging step left only sequences differing at positions −1, +1 and +2, relative to the m1A site
Investigations into the effect of m1A were mostly concerned with the application in structural probing in vitro [1], and the replication of the HIV genome in vivo, which strongly relies on Reverse Transcription (RT)-arrest induced by m1A58 of the HIV-primer tRNALys3 [32,33,34]
Summary
Several recent papers, including applications of different RNA-Seq protocols have created data containing mismatched nucleoside signals at sites known or postulated to contain m1A [13,14]. This strongly suggests that reverse transcriptase is capable of reading through this altered Watson–Crick face, thereby incorporating nonmatching nucleotides in the process and leaving unobtrusive traces of the m1A modification in cDNA data. We find a dependence on the type of the m1Apreceding nucleotide in the RNA template (i.e. to the 3 of m1A), whose nature correlates with misincorporation patterns These findings have important bearings for both areas: in structural probing, proper interpretation of RTarrest assays of DMS treated RNA should include the no-
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