title: Functionality ofIn vitroReconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles.
abstract: The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming.In vitroreconstituted ribonucleoprotein complexes from theLactococcus lactisgroup II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. TheSinorhizobium melilotigroup II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3' end to initiate reverse transcription. We used anEscherichia coliexpression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified componentsin vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.
authors.affiliation: Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Molina-Sánchez
authors.affiliation: Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Fernando M
authors.initials: FM
authors.lastname: García-Rodríguez
authors.affiliation: Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Toro
keywords: DNA endonuclease; catalytic RNAs; intron-encoded protein; maturase; reverse splicing; reverse transcriptase; ribonucleoprotein particles