Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

Abstract

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5 and 3 extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2-5 phosphodiester bond, and tandem, head-to-tail intron copies.Group II introns are large catalytic RNAs with a conserved secondary structure consisting of six domains arranged around a central wheel (1). Group II introns may self-splice via several different pathways (Fig. 1). The main splicing pathway in group II introns involves two sequential trans-esterification reactions. In the first, the 2Ј-hydroxyl of a bulged adenosine near the end of the intron, in domain VI, triggers a nucleophilic attack on the 5Ј splice site. In the second reaction, the 2Ј-5Ј branched product is released as a lariat when the free 3Ј-OH at the end of the 5Ј exon attacks the 3Ј splice junction joining the two exons (2-5). In ai5␥, a yeast mitochondrial group IIB intron, the functional group II intron branch site consists of the bulged adenosine, the wobble pairs (G-U) flanking this residue and, possibly, the purine-enriched sequence in domain VI on the strand opposite the bulged nucleotide (6 -8). Recent studies have reported the presence, within domain I of group IIB introns, of a branch site receptor known as a coordination loop (9), which is required for trans-esterification (8), but these findings have been called into question (10). This coordination loop has been identified as a key element for exon ligation, as it positions the exon and intron binding sequences, the EBS3-IBS3 base pair and the EBS1-IBS1 helix, in an appropriate conformation for reaction (9). There is also an alternative splicing pathway, the first step of which involves hydrolysis. The ligated exons and linear intron are then generated in a standard second step (11-13). An additional excision reaction, in which the intron is excised as a putative circle, has also been proposed for some group II introns (14 -16). In this case, prior release of the 3Ј exon from the precursor molecules, presumably via a transsplicing mechanism, seems to be required for the generation of a putative 2Ј-5Ј ligated circular intron. The origin and function of such intron circles in vivo remains unknown.RmInt1 is a mobile subclass IIB3 intron (17). It has also been assigned to phylogenetic bacterial class D (17, 18) on the basis o...

The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway

“…As previously observed (Li-Pook-Than and Bonen 2006; Molina- Sánchez et al 2006Sánchez et al , 2011Dalby and Bonen 2013), we detected some perfect intron RNA circles harboring extra nucleotides at the splice junction (Supplemental Table S1). The majority of the intron RNA circles for Ll.LtrBLtrAMat − (8/9) and Ll.LtrB-ΔA (30/31) harbored an extra C at the junction.…”

“…RmInt1 seems to share the same circularization pathway as Ll.LtrB as a significant portion of the circular introns released from a maturase mutant also harbors an extra C at the junction (Molina-Sánchez et al 2006), while removal of the branch point prevents lariat formation and leads to the exclusive release of intron circles also harboring an extra C (Molina-Sánchez et al 2011). It was recently proposed that the presence of an extra C at the splice junction of in vitro spliced RmInt1 is due to 3 ′ splice site misrecognition resulting in the inclusion of the first nucleotide of the 3 ′ exon (Chillón et al 2014).…”

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

“…RT-PCR was performed as previously described ([30]; Figure S2A). First-strand cDNA synthesis was performed with 8 µg of total cellular RNA, 25 pmol of the Ect1-specific primer (5′-CACCTGCTCGGATCTCGTC-3′) and SuperScript II RNase H − reverse transcriptase (Invitrogen), as indicated in the manufacturer’s protocol.…”

Localization of a bacterial group II intron-encoded protein in human cells

“…RNA was isolated with the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RT-PCR was performed as previously described 32 . First-strand cDNA synthesis was performed with 1 μg of total RNA, 25 pmol of the PE1 specific primer (5′-TCCTCGACGCTCATACG-3′; complementary to nt 461-nt 477 sequence from the 5′ end of RmInt1) and superscript II RNase H − reverse transcriptase (Invitrogen), as indicated in the manufacturer’s protocol.…”