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External guide sequences (EGSs) have successfully been used to inhibit expression of target genes at the posttranscriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS) libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from Francisella tularensis, a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested in vitro and in vivo. EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage in vitro. Expression of stem EGS73 and EGS155 in Escherichia coli resulted in significant reduction of the mglB mRNA level coded for the F. tularensis mglB gene inserted in those cells. [ABSTRACT FROM AUTHOR]

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using theTetrahymenaribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to theTetrahymenamodel ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomyceteDidymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3′ splice site (side reaction) without affecting self-splicing[Haugen, P., Andreassen, M., Birgisdottir,Å.B.&Johansen, S.D. (2004)Eur. J. Biochem.271, 1015–1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3′ splice site, for application in group I ribozyme-mediatedtrans-splicing of RNA. The improved ribozyme catalyses bothcis-splicing andtrans-splicingin vitroof a humanglycosylasparaginasemRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis. [ABSTRACT FROM AUTHOR]