Circularization pathway of a bacterial group II intron

Abstract

Group II introns are large RNA enzymes that can excise as lariats, circles or in a linear form through branching, circularization or hydrolysis, respectively. Branching is by far the main and most studied splicing pathway while circularization was mostly overlooked. We previously showed that removal of the branch point A residue from Ll.LtrB, the group II intron from Lactococcus lactis, exclusively leads to circularization. However, the majority of the released intron circles harbored an additional C residue of unknown origin at the splice junction. Here, we exploited the Ll.LtrB-ΔA mutant to study the circularization pathway of bacterial group II introns in vivo. We demonstrated that the non-encoded C residue, present at the intron circle splice junction, corresponds to the first nt of exon 2. Intron circularization intermediates, harboring the first 2 or 3 nts of exon 2, were found to accumulate showing that branch point removal leads to 3′ splice site misrecognition. Traces of properly ligated exons were also detected functionally confirming that a small proportion of Ll.LtrB-ΔA circularizes accurately. Overall, our data provide the first detailed molecular analysis of the group II intron circularization pathway and suggests that circularization is a conserved splicing pathway in bacteria.

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

“…During the formation of circRNAs either 2 0 -5 0 or 3 0 -5 0 linkages have been detected. 8,9 The resulting molecular "rings" or RNA circles resist degradation by exoribonucleases that require free termini and/or may have increased melting point in comparison to linear nucleic acid molecules. Circularization of RNA molecules may thus drastically influence the structure and/or shape of these molecules, presumably reflecting structural constraints brought about by circularization.…”

Bacterial group II introns generate genetic diversity by circularization and trans-splicing from a population of intron-invaded mRNAs

“…To study the splicing pathway leading to the incorporation of additional nucleotides at the splice junction of group II intron circles [ 17 ] we performed an RT-PCR reaction across the Ll.LtrB-ΔLtrA+LtrA lariat and circle splice junctions ( Fig 2 ) [ 17 , 18 ]. We cloned and sequenced the amplicons located in the faint smear above the RT-PCR band that corresponds to perfect lariat and circle splice junctions ( Fig 2C ).…”

“…We recently unveiled and characterized at the molecular level the circularization pathway of Ll.LtrB, the model group II intron, from the gram-positive bacterium Lactococcus lactis [ 17 , 18 ]. Our work showed that the intron excises simultaneously through the branching and circularization pathways in vivo leading to the accumulation of both intron lariats and circles respectively.…”

Recent horizontal transfer, functional adaptation and dissemination of a bacterial group II intron

“…To determine whether Ef.PcfG is functional in its native environment, we first assessed its ability to splice in vivo by looking for both released intron and ligated exons by RT-PCR on E. faecalis (SF24397ΔpEF1071) total RNA extracts (Fig. 2a ) [ 35 , 36 ]. Sequencing of the major amplicons for both ligated exons and released intron showed that they correspond respectively to accurately joined pcfG exons (1587 bp) (Fig.…”

“…To evaluate the splicing efficiency of our various intron constructs, we performed poisoned primer extension assays (Fig. 4a ) [ 35 , 39 ]. This assay compares the ratio of ligated exons to precursor mRNA from total RNA extracts.…”