Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA

Abstract

Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.

Towards Reverse Transcription with an Expanded Genetic Alphabet

“…In this study, we evaluate, for the first time, the rtc reaction based on the TPT3:NaM base pair (Figure A). We aimed to identify those reverse transcriptases (RTs) that are efficiently blocked at the site of the unnatural nucleotide, and allow quantification of rTPT3 and rNaM in RNA as well as the identification of others which exhibit the first signs of compatibility with the UBP (Figure B). In detail, we employed four commercially available retroviral‐derived RTs: avian myeloblastosis virus (AMV) RT, moloney murine leukemia virus (MMLV) RT, SuperScript II (SS II) RT, and SuperScript IV (SS IV) RT; the last two represent genetically engineered MMLV RT variants.…”

“…In detail, we employed four commercially available retroviral‐derived RTs: avian myeloblastosis virus (AMV) RT, moloney murine leukemia virus (MMLV) RT, SuperScript II (SS II) RT, and SuperScript IV (SS IV) RT; the last two represent genetically engineered MMLV RT variants. Retroviral RTs naturally possess low fidelity and processivity because the introduction of mutations is essential for retroviruses to evade the host defense and might therefore be suitable for processing the UBP system. We further used a novel engineered Taq DNA polymerase with RT activity: Volcano2G (V2G) .…”

“…Rtc from unmodified RNA will result in excess primer band and full‐length cDNA product band (Figure A, lane a); rtc from RNA modified to 100 % with rTPT3 at position 34 by using only canonical TPs will result in pausing before or at this position if the RT is not able to process the UBP (Figure A, lane b). Alternatively, a mutagenic read through (misincorporation or skipping of the UBP) is imaginable and a mixture of both cases …”

Anomalous reverse transcription through chemical modifications in polyadenosine stretches

“…12 ). Other steps with potential bias include the synthesis of the RNA by in vitro transcription with T7 RNA polymerase, which is known to 'slip' on repeated sequences 20 ; similar slippage or biased termination of reverse transcriptases in the RTase step after chemical modification [32][33] ; the ligation of adapters onto the resulting cDNAs, which are known to have sequence biases 22,30,34 ; Illumina sequencing-by-synthesis of cDNAs used, which is also known to have sequence biases 29 ; or the bioinformatic workup of these sequencing data into inferred chemical modification profiles. 29,35 We first sought to test if the poly(A) SHAPE and DMS signatures were due to the many possible biases occurring any steps downstream of reverse transcription, by carrying out the simplest method of reading out the cDNA products, capillary electrophoresis with fluorescently labeled primers.…”

The evolution of RNA structural probing methods: From gels to next‐generation sequencing

“…Thus, depending on the buffer conditions, SuperScript enzymes can be adapted to both read out strategies. Interestingly, a number of groups have also begun using TGIRT enzyme for mutational profiling purposes (Sexton, Wang, Rutenberg-Schoenberg, & Simon, 2017;Zubradt et al, 2017). In the case of DMS (methylation at A/C residues), a direct comparison between SSII (Mn 2+ buffer) and TGIRT revealed that reproducible and robust results were best obtained with TGIRT (Zubradt et al, 2017).…”