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Group I intron ribozymes can repair mutated mRNAs by replacing the 3′-terminal portion of the mRNA with their own 3′-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5′-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 106 ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.

Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5′- or 3′-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This 'group I spliceozyme' functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy. © 2014 Amini et al.

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell. © 2014 Olson et al.