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.

SUBMITTER: Doring J 

PROVIDER: S-EPMC5399748 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature