Evaluating group I intron catalytic efficiency in mammalian cells

Recent reports have demonstrated that the group I ribozyme from Tetrahymena thermophila can perform trans-splicing reactions to repair mutant RNAs. Fo r therapeutic use, such ribozymes must function efficiently when transcribe d from genes delivered to human cells, yet it is unclear how group I splici ng reactions are influenced by intracellular expression of the ribozyme. He re we evaluate the self-splicing efficiency of group I introns from transcr ipts expressed by RNA polymerase II in human cells to directly measure ribo zyme catalysis in a therapeutically relevant setting, Intron-containing exp ression cassettes were transfected into a human cell line, and RNA transcri pts were analyzed for intron removal. The percentage of transcripts that un derwent self-splicing ranged from 0 to 50%, depending on the construct bein g tested. Thus, self-splicing activity is supported in the mammalian cellul ar environment, However, we find that the extent of self-splicing is greatl y influenced by sequences flanking the intron and presumably reflects diffe rences in the intron's ability to fold into an active conformation inside t he cell. In support of this hypothesis, we show that the ability of the int ron to fold and self-splice from cellular transcripts in vitro correlates w ell with the catalytic efficiency observed from the same transcripts expres sed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozyme s, The self-splicing system that we describe should facilitate these effort s as well as aid in efforts at enhancing in vivo ribozyme activity for vari ous applications of RNA repair.