An RNA secondary structure bias for non-homologous reverse transcriptase-mediated deletions in vivo.
Murine leukemia viruses harboring an internal ribosome entry site (IRES)-directed translational cassette are able to replicate, but undergo loss of heterologous sequences upon continued passage. While complete loss of heterologous sequences is favored when these are flanked by a direct repeat, deletion mutants with junction sites within the heterologous cassette may also be retrieved, in particular from vectors without flanking repeats. Such deletion mutants were here used to investigate determinants of reverse transcriptase-mediated non-homologous recombination. Based upon previous structural analysis the individual recombination sites within the IRES could be assigned to either base-paired or unpaired regions of RNA. This assignment showed a significant bias (P = 0.000082) towards recombination within unpaired regions of the IRES. We propose that the events observed in this in vivo system result from template switching during first-strand cDNA synthesis and that the choice of acceptor sites for non-homologous recombination are guided by non-paired regions. Our results may have implications for recombination events taking place within structured regions of retroviral RNA genomes, especially in the absence of longer stretches of sequence similarity.
Duch
Mogens
M
Department of Molecular Biology, University of Aarhus, C.F. Mollers Allé, Building 130, DK-8000 Aarhus, Denmark.
Carrasco
Maria L
ML
Jespersen
Thomas
T
Aagaard
Lars
L
Pedersen
Finn Skou
FS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011995:Recombination, Genetic; D038621:Regulatory Sequences, Ribonucleic Acid; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014779:Virus Replication
D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D012194:RNA-Directed DNA Polymerase