Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

Abstract

Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication.

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA

“…2 C ). In contrast, we did not observe bRT-dependent termination at the RNA-cDNA junction, which would be expected if there were a branched intermediate resulting from cDNA priming by the 2′-OH of sp A56 ( 14 – 16 ).…”

Spontaneous advent of genetic diversity in RNA populations through multiple recombination mechanisms

“…This would result in the fragments we observed. Moreover, some RNase H-deficient M-MLV reverse transcriptases frequently cause mutations and/or deletions around the branch point (Döring and Hurek 2017), which is consistent with our detection of the slight variants of R16 and H13 (Tables 1, 2). The observation that the γ/γ ′ ′ ′ ′ ′ products appear prior to any other product in both self-incubations (Fig.…”

“…Approximately 89% of products from each reaction fit this pattern. While many of these could be the result of fragmentation during the preparation for the HTS analysis, or from contamination of the starting material during gel purification (despite the many countermeasures em-ployed), we propose that a more likely explanation for their abundance is the tendency of RNase-H deficient reverse transcriptases such as Superscript IV (which we used to obtain the data in Tables 1, 2) to generate both truncated products at a 2 ′ -5 ′ -3 ′ branched junction and full-length cDNA upon reading through the 3 ′ ,5 ′ side of such branches (Döring and Hurek 2017). This would result in the fragments we observed.…”

Evaluation of various RNA-seq approaches for identification of gene outrons in the flatworm Opisthorchis felineus

“…1, c). It is known that MMLV reverse transcriptase lacking RNase H activity, when primed from the 2′arm, is able to quite frequently bypass the branchpoint with the introduction of singlemismatch errors and successfully reverse the 5′segment (Bitton et al, 2014;Döring, Hurek, 2017). Among the expected drawbacks of the method are the reduced efficiency of such reverse tran scription, the accumulation of products of mispriming, and the underrepresentation of the 5′regions of outrons due to a frequent accidental termination of the first strand cDNA syn thesis.…”