RNA/protein interactions during group II intron splicing and toward group II intron targeting in mammalian cells

textGroup II introns are both catalytic RNAs and retrotransposable elements. Group II intron-encoded proteins (IEPs) have maturase activity, which promotes intron RNA splicing, and reverse transcriptase activity, which functions in intron mobility. Previous studies of the Lactococcus lactis Ll.LtrB intron suggested a model in which its IEP binds first to a high-affinity binding site in intron subdomain DIVa and then makes additional contacts with the conserved catalytic core to stabilize the active RNA structure. In the absence of DIVa, the IEP promotes residual splicing by binding directly to the catalytic core. I developed E. coli genetic assays to detect in vivo splicing of the Ll.LtrB intron and identify regions in the IEP essential for interacting with different parts of the intron RNA. Mutational and biochemical analysis combined with three-dimensional structural modeling support the hypothesis that the extended N-terminal finger region of LtrA is involved in high-affinity binding of DIVa, possibly forming a binding pocket in combination with parts of the thumb domain (domain X), while other regions of the RT and X domains are potentially involved in binding the catalytic core. The Ll.LtrB intron works very efficiently for gene targeting in bacteria, and it is desirable to target mammalian genomes, for which efficient means of manipulation are lacking. I developed an expression system to produce Ll.LtrB intron RNA and IEP in cultured human cells and found that the expressed intron splices in vivo. I also explored different methods for introducing RNPs into cells, including electroporation and injection, and was able to detect a chromosomal targeting event with RNP electroporation. With improvements in targeting efficiency, group II introns would be generally useful for functional genomics and gene therapy.Institute for Cellular and Molecular Biolog