An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells.
The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme. The resulting cleavage products are subsequently joined together by an endogenous ligase. We demonstrate the potential of using this strategy for repairing RNA in higher organisms by expressing the enzyme in mouse cells. Reporter target mRNAs modified with 17-nucleotide introns, flanked by sequences capable of forming BHB structures in cis, were expressed in mouse cells. RNA molecules that can form BHB substrates in trans with targeted mRNAs were also designed. Co-transfection of mouse cells with plasmids expressing these RNAs and the MJ endonuclease led to formation of RNA chimeras in which the target and exogenous RNA were recombined across the BHB. This technology is not limited to mRNA, but could in principle be used to destroy, modify or restore the function of a vast repertoire of RNA species or to join selectable tags to target RNAs.
Istituto di Biologia Cellulare, CNR, Campus A. Buzzati-Traverso, Via E. Ramarini, 32, 00016 Monterotondo Scalo, RM, Italy.
Giancarlo
G
Deidda
Nicoletta
N
Rossi
Glauco P
GP
Tocchini-Valentini
D016475:3T3 Cells; D000818:Animals; D004722:Endoribonucleases / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D017017:Methanococcus / Q000201:enzymology*; D051379:Mice; D009154:Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D020040:Trans-Splicing / Q000235:genetics*
D012333:RNA, Messenger; D004722:Endoribonucleases