Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3 splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.Group II introns are large catalytic RNAs with a conserved secondary structure consisting of six domains, one of which (dIV) may incorporate the coding sequence of a reverse transcriptase (RT) 3 (1). Although some group II introns self-splice in vitro, this reaction requires nonphysiological conditions, and in vivo, proteins are required to fold the intron RNA into a catalytically active structure. Group II intron-encoded proteins (IEPs) promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein (RNP) complex (2-4). The IEPs have two conserved domains, an N-terminal RT domain and domain X, a putative RNA-binding domain associated with maturase (RNA splicing) activity. In some cases the IEP also includes a C-terminal DNA-binding and a DNAendonuclease domain (5).Group II introns splice typically by the same two sequential transesterification reactions used in nuclear mRNA splicing (1). In a first step, the 2Ј-OH group of a branch point nucleotide residue, usually a bulged adenosine in dVI, attacks the 5Ј splice junction resulting in cleavage of the 5Ј exon and the formation of an intron-3Ј exon-branched lariat intermediate. The released 5Ј exon remains associated to the intron via base pairing of the intron-binding sites (IBS1 and IBS2) to the exon-binding sites (EBS1 and EBS2) located in domain dI. In the second step, the free 3Ј-OH of the 5Ј exon attacks the 3Ј splice junction leading to the release of the intron lariat and the ligation of the 5Ј and 3Ј exons. There also exists an alternate pathway, in which the first splicing step is initiated by a nucleophilic attack of water or an OH Ϫ ion, resulting in the formation of a linear intron-3Ј exon intermediate that subsequently participates in a normal second step reaction. This hydrolytic pathway is observable in vitro and has been shown to be used in vivo by yeast mitochondrial introns carrying branch-site mutations (6). Moreover, even though most of the plant chloroplast group II introns follow the typical lariat-generating pathway, hydrolytic splicing has been reported for intron trnV, which lacks the conserved bulged A in dVI, whereas both the hydrolytic and b...
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