Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.Group II introns are ribozymes and mobile retroelements consisting of a highly structured RNA organized into six distinct domains (dI to dVI). They have a multifunctional intron-encoded protein (IEP) ORF in dIV, encoding a reverse transcriptase-maturase that is required in vivo for the folding of the intron RNA into a catalytically active structure [1]. Group II introns splice via a lariat intermediate, in a mechanism similar to that of spliceosomal introns, via two sequential transesterification reactions [1]. In the first step, the 2'-OH group of a branch-point nucleotide residue, usually a bulged adenosine in dVI, attacks the 5' splice junction, resulting in cleavage of the 5' exon and the formation of an intron-3' exon branched lariat intermediate. The released 5' exon remains associated with the intron via base pairing of the intron binding sites (IBS1 and IBS2) to the exon binding sites (EBS1 and EBS2) located in ribozyme domain dI [2]. In the second step, the free 3'-OH of the 5' exon attacks the 3' splice junction, leading to the release of the intron lariat and the ligation of the 5' and 3' exons. Recognition of the 3'exon involves two additional base-pair interactions: the first involves the formation of a single tertiary base-pair between the last position of the intron (γ') and an intron