RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.
Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.
Fiskaa
Tonje
T
Department of Molecular Biotechnology, RNA Research group, Institute of Medical Biology, University of Tromsø, Norway.
Lundblad
Eirik W
EW
Henriksen
Jørn R
JR
Johansen
Steinar D
SD
Einvik
Christer
C
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D007438:Introns; D008969:Molecular Sequence Data; D009235:Myxomycetes / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D013768:Tetrahymena / Q000166:cytology / Q000235:genetics; D020040:Trans-Splicing; D043323:alpha-Mannosidase / Q000235:genetics*
C116167:GIR1 ribozyme; D016337:RNA, Catalytic; D012333:RNA, Messenger; D043323:alpha-Mannosidase