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An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells

Nature Biotechnology volume 21, pages 1499–1504 (2003)Cite this article

Abstract

The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme1. 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.

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Figure 1: Scheme of BHB insertions in reporter mRNAs.
Figure 2
Figure 3: Repair of a defective firefly luciferase mRNA by BHB-mediated trans-splicing.
Figure 4: Production of a Crat-EGFP mRNA by BHB-mediated trans-splicing.

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Acknowledgements

We thank R.H. Haselkorn for continuous encouragement and help, E.P. Geiduschek, D. Schlessinger and J.E. Dahlberg for valuable comments, A. Ferrara for secretarial assistance and G. Di Franco for technical assistance. Financial support was provided by the Programma Biomolecole per la Salute Umana MURST-CNR, FIRB Progetto di Ricerca Negoziale MIUR, Progetto Genomica Funzionale MIUR-CNR, Progetto Strategico Tecnologie di base della post-genomica CNR, Progetto Strategico Biotecnologie MURST-CNR, Progetto Strategico Genetica Molecolare MURST and EUMORPHIA European Network of Excellence.

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  1. Istituto di Biologia Cellulare, CNR, Campus “A. Buzzati-Traverso,” Via E. Ramarini, 32, Monterotondo Scalo, 00016, RM, Italy

    Giancarlo Deidda, Nicoletta Rossi & Glauco P Tocchini-Valentini

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  1. Giancarlo Deidda
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  2. Nicoletta Rossi
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Correspondence to Glauco P Tocchini-Valentini.

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Deidda, G., Rossi, N. & Tocchini-Valentini, G. An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells. Nat Biotechnol 21, 1499–1504 (2003). https://doi.org/10.1038/nbt908

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