We demonstrate that a group I intron-derived ribozyme from the opportunistic pathogen Pneumocystis carinii can bind an RNA in trans and excise from within it an internal segment, resulting in the splicing of the remaining ends of the RNA back together (the trans excision-splicing reaction). The reaction is intramolecular with regard to substrate. The ribozyme targets its substrate by base pairing with two or three noncontiguous regions on the substrate, and the reaction occurs through a nucleotide cofactor independent mechanism. The excised segment can be as long as 28 nucleotides, or more, and as little as one nucleotide. The potential usefulness of this reaction is demonstrated by engineering a ribozyme that excises the triplet-repeat expansion region from a truncated myotonic dystrophy protein kinase transcript mimic in vitro.
We previously reported that a group I intron-derived ribozyme can catalyze the excision of targeted sequences from within RNAs in vitro and that dissociation of the bridge-3' exon intermediate between the two reaction steps is a significant contributing factor to low product yields. We now analyze the effects of increasing the length, and thus the strength, of helices P9.0 and P10, which occur between the ribozyme and the bridge-3' exon region of the substrate, on this trans excision-splicing reaction. Using substrates where lengthy targeted regions are excised, these modifications can significantly increase product yields, specifically by enhancing the second reaction step. A threshold for product formation is obtained, however, at around five base pairs for P10 and eight base pairs for P9.0. Nevertheless, elongating P9.0 appears to be the more effective strategy, as both substrate binding and the rate of the second reaction step increase. In addition, P10 is required when P9.0 is not elongated. Also, a strong P9.0 helix cannot replace a weaker P10 helix, indicating that P9.0 and P10 play somewhat distinct roles in the reaction. We also show that second-step inhibition stems from the formation of an extended P1 helix (P1ex), consisting of as little as a single Watson-Crick base pair, as well as the mere presence of substrate nucleosides immediately downstream from P10. Both of these inhibitory components can be overcome by utilizing P9.0 and P10 elongated ribozymes. This work sets forth an initial framework for rationally designing more effective trans excision-splicing ribozymes.
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