title: Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells.
abstract: ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biologia Cellulare e Neurobiologia, European Mouse Mutant Archive, Monterotondo, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Putti
authors.forename: Patrizia
authors.initials: P
authors.lastname: Calandra
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Rossi
authors.forename: Daniela
authors.initials: D
authors.lastname: Scarabino
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Deidda
authors.forename: Glauco P
authors.initials: GP
authors.lastname: Tocchini-Valentini
mesh_terms: D001105:Archaea / Q000201:enzymology* / Q000235:genetics*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D016133:Polymerase Chain Reaction; D012326:RNA Splicing / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D004720:Endonucleases
keywords: Methanocaldococcus jannaschii; bulge-helix-bulge