In vivo repair of viral transcripts following cleavage by a cis hairpin ribozyme
The hairpin ribozyme is a catalytic RNA that can be engineered to cleave any RNA with the sequence 5' GUC 3'. Because of this capacity to cleave targeted RNA, the hairpin ribozyme has the potential to down-regulate any particular gene of interest either in vitro or in vivo. Previous work from our lab showed that a cis- cleaving hairpin cassette inserted into the cauliflower mosaic virus genome could temporarily attenuate viral symptoms in Brassica campestris rapa. However, viral symptoms did eventually appear, suggesting that the virus was either outcompeting the relatively slow ribozyme cleavage or was repairing its genome. Further studies revealed deletions in several ribozyme DNA sequences from later time periods postinoculation, possibly indicating in vivo viral deletion of ribozyme sequences. Because these deletions always appeared immediately 3' of the cleavage site, they might have occurred at the RNA level. Base substitutions also were found in 6 of 26 ribozyme sequences examined. Since CaMV uses reverse transcriptase to replicate, an enzyme noteworthy for its tendency to produce a relatively high frequency of sequence alterations (including base substitutions, deletions, and recombinations), these mutations may be the result of reverse transcription. We suggest the possibility that reverse transcriptase might have produced the ribozyme deletions by a novel mechanism of "jumping" from secondary RNA structure in the 3' fragment to the cleaved end of the 5' fragment.