Fig 1.
Group II intron splicing pathways in bacterial cells.
(A) Branching pathway. Following transcription of the intron-interrupted gene, the 2´-OH residue of the branch-point A nucleotide initiates the first nucleophilic attack at the E1-intron 5′ splice junction (step 1). This transesterification reaction connects the 5´ end of the intron to the 2´-OH residue of the branch point A and releases E1 that remains associated to the intron through EBS1/2-IBS1/2 base pairing interactions (vertical lines). The liberated 3´-OH at the end of E1 then initiates the second nucleophilic attack at the intron-E2 3´ splice junction, ligating the two exons (E1-E2) and releasing the intron as a lariat or branched structure (step 2). (B) Hydrolytic pathway. The first transesterification reaction at the 5′ splice junction is initiated by an external nucleophile (H2O / OH-) leading to the release of E1 (step 1). The liberated 3´-OH at the end of E1 then initiates the second nucleophilic attack at the 3´ splice junction releasing the intron in linear form while ligating the two exons (E1-E2) (step 2). (C) Circularization pathway. The first nucleophilic attack takes place at the intron-E2 3´ splice junction and is initiated by the 3´-OH of a free E1 (step 1), generating ligated exons (E1-E2) and a circularization splicing intermediate where the 5′ end of the linear intron is still attached to E1. This reaction is not always accurate generating a series of circularization intermediates where the first nucleotides (nts) of E2 are still attached to the intron 3′ end [4]. Next, the 2´-OH of the last intron residue initiates the second nucleophilic reaction at the E1-intron 5´ splice junction resulting in the release of perfect head to tail intron circles and free E1 (step 2). A potential source of free E1 is the spliced exon reopening reaction (panel A and B, SER) where linear introns and lariats can recognize and hydrolyze ligated exons (E1-E2) at the splice junction.
Table 1.
Primers.
Fig 2.
Splicing efficiency and detection of circularization intermediates for various Ll.LtrB constructs.
(A) Position of the 6 mutations (arrows) engineered around the -138 processing site (black arrowhead) in DIVb of Ll.LtrB-WT are shown (Ll.LtrB-Mut-138). The position of DIVb relative to the overall Ll.LtrB secondary structure (DI-DVI) is also depicted (dashed area). (B) The splicing efficiency of Ll.LtrB variants expressed from the pDL-P232 plasmid was assessed by the poisoned primer extension assay [4, 21]. This assay monitors intron splicing efficiency by comparing the relative abundance of precursor mRNAs (53 nts) and ligated exons (WT, Mut-138 and AU-rich: 51 nts; Mut-IBS2 and IBS2-EBS2 swap: 44 nts; GC-rich: 38 nts; IBS1-EBS1 swap: 37 nts) from total RNA extracts. (C) The poisoned primer extension reactions were run on a 8% PAGE 8M urea gel and splicing efficiency (Splicing efficiency %) was calculated as the relative intensity of the ligated exons (LE) and precursor (P) bands (LE / (P + LE)). (D) Free intron 3′ ends were identified from L. lactis total RNA extracts by rapid amplification of cDNA 3′ ends (3′ RACE) [4]. The PCR reactions were run on a 2% agarose gel (E). Amplicons corresponding to the circularization intermediates (~500 nts) and the -138 processing site (~350 nts) were all recovered and sequenced. Chromatograms of the circularization intermediates (~500 nts) for WT (F) and Mut-138 (G) are shown.
Fig 3.
Circle to lariat ratios of various Ll.LtrB constructs at different growth temperatures.
(A) Scheme of the combined Ll.LtrB circularization and branching splicing pathways in L. lactis. Position of the primers used to amplify the excised intron circle (144 bp) and lariat (138 bp) splice junctions by RT-PCR is depicted (open arrows). (B) RT-PCR perfomed on total RNA extracts from L. lactis grown at various temperatures (20°C to 40°C) and expressing different Ll.LtrB variants from the pDL-P232 plasmid. The amplicons were labelled at the 5′ end with 32P and ran on 8% denaturing PAGE gels [5]. The IBS1/EBS1 and IBS2/EBS2 base pairing interactions between the intron (EBS1/2) and the 3′ end of E1 (IBS1/2) are shown along with the position of the 5′ splice site (black arrowhead). Two or three hydrogen bonds between complementary nts are denoted by a dotted or solid line respectively. The circle to lariat (circle:lariat) ratios (circle / (circle + lariat):lariat / (lariat + circle) are displayed under each lane.
Fig 4.
Circle to lariat ratios of three bacterial group II introns in various cellular environments.
RT-PCR amplification of excised intron junctions was perfomed on total RNA extracts of bacterial cells expressing intron-interrupted relaxase genes in their native environments (A) (ltrB gene from L. lactis strain NZ9800 (Ll.LtrB intron); pcfG gene from Enterococcus faecalis strain SF24397 (Ef.PcfG intron); rlxA gene from L. lactis strain LMG9447 (Ll.RlxA intron)), and also from the P23 constitutive promoter in L. lactis NZ9800ΔltrB (B), in L. lactis LMG9447 (C) and in E. faecalis JH2-2 (D). The branch point variant of Ll.LtrB (Ll.LtrB-ΔA) was also expressed from the P23 constitutive promoter in the three host environments (B-D). The circle to lariat (circle:lariat) ratios (circle / (circle + lariat):lariat / (lariat + circle) are displayed under each lane. The lower band in the Ll.RlxA lane (panel A) was sequenced and corresponds to the non-specific amplification of the unspliced precursor mRNA (intron 3′ end-Exon 2). N/D (not detected): no intron circle and lariat detected. N/A (not applicable): Ll.LtrB-ΔA splices exclusively through circularization.
Fig 5.
Circle to lariat ratios of three bacterial group II introns expressed at different levels in L. lactis.
RT-PCR amplification of excised intron junctions was perfomed on total RNA extracts of the NZ3900 L. lactis strain expressing three intron-interrupted relaxase genes (ltrB gene from L. lactis strain NZ9800 (Ll.LtrB intron); pcfG gene from Enterococcus faecalis strain SF24397 (Ef.PcfG intron); rlxA gene from L. lactis strain LMG9447 (Ll.RlxA intron)) from the strong nisin-inducible promoter (Pnis). The circle to lariat (circle:lariat) ratios (circle / (circle + lariat):lariat / (lariat + circle) are displayed under each lane. N/D (not detected): no intron circle and lariat detected. The lower band in the Ll.RlxA lane was sequenced and corresponds to the non-specific amplification of the unspliced precursor mRNA (intron 3′ end-Exon 2). Induction conditions: non-induced overnight culture, No Nisin ON; nisin-induced overnight culture, Nisin ON; 7h exponential growth nisin-induced culture, Nisin 7h; nisin-induced overnight culture followed by 7h exponential growth nisin-induced culture, Nisin ON + 7h.