Fig 1.
Group II intron splicing pathways.
(a) Branching pathway. Following transcription of the interrupted gene, the 2´-OH residue of the branch-point nucleotide (A) initiates the first nucleophilic attack at the exon 1-intron junction (step 1). This transesterification reaction connects the 5´ end of the intron to the branch point and releases exon 1 that remains associated to the intron through base pairing interactions (EBS-IBS interactions) (vertical lines). The liberated 3´-OH at the end of exon 1 then initiates a second nucleophilic attack at the intron-exon 2 junction (step 2), ligating the two exons and releasing the intron as a lariat. (b) Hydrolytic pathway. A hydroxyl ion or a water molecule initiates the first nucleophilic attack at the exon 1-intron junction (step 1). The second nucleophilic attack at the intron-exon 2 junction is initiated by the liberated 3´-OH at the end of exon 1 (step 2) which ligates the two exons and releases a linear intron. (c) Circularization pathway. The first nucleophilic attack takes place at the intron-exon 2 junction and is initiated by the 3´-OH of a free exon 1 (step 1) generating ligated exons and a circularization intermediate where the linear intron is still attached to exon 1. Next, the 2´-OH of the last intron residue is thought to initiate the second nucleophilic reaction at the exon 1-intron junction (step 2) resulting in intron circularization and the release of free exon 1. A potential source of free exon 1 is the spliced exon reopening (SER) reaction where both excised lariats and linear introns can recognize and hydrolyze ligated exons at the splice junction. To explain the presence of additional nts at the splice junction of intron circles, the external nucleophilic attack pathway (d) was previously proposed [17, 19]. The 3’OH residue of a block of external nts (grey box) attacks the exon 1-intron junction, ligating it to the intron 5’ end while concurrently displacing exon 1 (step 1). The 3’OH at the end of exon 1 then attacks the intron-exon 2 junction releasing ligated exons and a linear intron harboring external nts at its 5’ end (step 2). The third transesterification reaction is initiated by the 2’-OH of the last intron residue (step 3). The position of this final nucleophilic attack thus dictates how many additional nts are incorporated at the junction of intron circles.
Fig 2.
Detection of mRNA fragments at the splice junction of excised intron RNA circles.
(a) Various Ll.LtrB constructs used in this study where the LtrA protein is provided either in trans (Ll.LtrB-ΔLtrA+LtrA, Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA) or in cis (Ll.LtrB-WT) (b) Schematic of Ll.LtrB self-splicing. Position of the primers (open arrows)(S2 Table) used to amplify the splice junction of excised introns by RT-PCR is depicted (92 bp (lariat), 98 bp (circle) or >98 bp (circle harboring additional nts)). (c) RT-PCR amplifications of intron splice junctions. Amplifications were performed on total RNA extracts from L. lactis (NZ9800ΔltrB) harboring different Ll.LtrB constructs expressed under the control of the P23 constitutive promoter (ΔLtrA+LtrA: pDL-P232-Ll.LtrB-ΔLtrA and pLE-P232-LtrA)(WT: pDL-P232-Ll.LtrB-WT)(EBS1/Mut: pDL-P232-Ll.LtrB-EBS1/Mut-ΔLtrA and pLE-P232-LtrA). The EBS1/Mut intron variant was shown to splice accurately and efficiently in vivo by RT-PCR amplifications of both released introns and ligated exons. Additional nts incorporated at the junction of intron circles are represented by a gray box.
Fig 3.
mRNA fragments identified at the splice junction of Ll.LtrB circles.
Additional nts are shown along with their flanking sequences (5’ flanking) (3’ flanking), their origin (Gene name) and frequency of identification between parentheses for Ll.LtrB-ΔLtrA+LtrA (a) and Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA (b) circles. The junctions between the additional nts and their flanking regions (/) as well as the IBS1- (yellow) and IBS2- (green) like sequences are denoted. The bolded nts represent residues from the IBS1- and IBS2-like sequences that can potentially base pair with the intron’s EBS1 and EBS2 sequences specified above. Sequences spanning two genes and including a short intergenic region are underlined. The genes in bold (alaS, enoA, S12/S7) were further studied for Ll.LtrB reverse splicing analyses and the detection of E1-mRNA and mRNA-mRNA chimeras (Figs 7 and 8).
Fig 4.
Logo representation of the consensus sequences (30 nts) around the 5’ and 3’ extremities of the mRNA fragments identified at intron circle splice junctions.
The EBS1-IBS1 and EBS2-IBS2 base pairing interactions for Ll.LtrB-ΔLtrA+LtrA and Ll.LtrB-WT (a) as well as Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA (d) are depicted. The consensus sequences are shown for Ll.LtrB-ΔLtrA+LtrA (Fig 3A)(b), Ll.LtrB-WT (S1 Fig)(c) and Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA (Fig 3B)(e).
Fig 5.
Models for the incorporation of mRNA fragments at the splice junction of intron RNA circles.
(a) External nucleophilic attack pathway [17, 19]. The Ll.LtrB group II intron recognizes, through base pairing interactions, an IBS1/2-like sequence (—|—) on an mRNA and guides the first nucleophilic attack induced by an hydroxyl ion or a water molecule downstream of the recognized sequence (step 1). Next, the 3’-OH of the processed mRNA induces a nucleophilic attack at the exon 1-intron splice junction resulting in its ligation to the 5’ end of the intron and the release of exon 1 (step 2). The 3’-OH of exon 1 is then free to initiate the second transesterification reaction at the intron-exon 2 splice junction, releasing ligated exons and a linear intron harboring a fragment of mRNA at its 5’ end (step 3). The final transesterifictaion reaction is induced at the intron 5’ end (a) or within the mRNA (b) by the 2’-OH of the last nt of the linear intron, just downstream from IBS1/2-like sequences (—|—), resulting in the release of either a head-to-tail circular intron (step 4a) or an intron circle harboring an mRNA fragment at its splice junction (step 4b). (b) Reverse splicing pathway. This pathway is initiated by the reverse splicing of an intron lariat within a non-cognate mRNA downstream of an IBS1/2-like sequence (—|—)(step 1). The 3’-OH of free exon 1 then attacks the phosphodiester bond at the 3’ splice site between the last nt of the intron and the 3’ segment of the mRNA (step 2). This generates a chimeric mRNA consisting of the ltrB-exon 1 (E1) linked to the 3’ segment of the mRNA (E1-mRNA) and a circularization intermediate where the linear intron is still attached to the 5’ segment of the mRNA. The third transesterifictaion reaction is induced at the intron 5’ end (a) or within the mRNA fragment (b) by the 2’-OH of the last residue of the linear intron, just downstream from IBS1/2-like sequences (—|—), resulting in the release of either a head-to-tail circular intron (step 3a) or an intron circle harboring an mRNA fragment at its splice junction (step 3b). The 3’ junction of reverse-spliced introns and the chimeric E1-mRNAs are unique splicing intermediates that distinguish both pathways (asterisks).
Fig 6.
Ll.LtrB reverse splicing within L. lactis mRNAs.
(a) Independent Ll.LtrB reverse splicing events were identified by total RNA-Seq for Ll.LtrB-ΔLtrA+LtrA, Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA and the control Ll.LtrB-ΔA-ΔLtrA+LtrA that exclusively splices through the circularization pathway and cannot reverse splice. The EBS1-IBS1 and EBS2-IBS2 base pairing interactions for Ll.LtrB-ΔLtrA+LtrA (b) and Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA (c) are depicted. Logo representations of the consensus sequences upstream (15 nts) and downstream (15 nts) from the intron reverse splice sites within the various L. lactis mRNAs are also shown.
Fig 7.
Detection of intermediates unique to the reverse splicing pathway: Ll.LtrB reverse-spliced within L. lactis mRNAs, E1-mRNA and mRNA-mRNA chimeras.
RT-PCR assays were performed to detect the 5’ and 3’ junctions of Ll.LtrB-ΔLtrA+LtrA and Ll.LtrB-ΔA-ΔLtrA+LtrA reverse splicing events within the enoA (red boxes)(a, b) and alaS (blue boxes)(e, f) mRNAs. Complete and dashed arrows indicate reverse splicing of Ll.LtrB lariats within strong (S) and weak (W) IBS1/2-like sequences (—|—) respectively. The strong (S)(10/11 nts)(large arrowhead) and weak (W)(7-9/11 nts)(small arrowhead) IBS1/2-like sequences invaded by reverse splicing are represented (c, g). The sites flanking the mRNA fragments (c, 167 nts and g, 304 nts) initially detected at intron circle splice junctions (Fig 3A) are indicated by open arrowheads. The Ll.LtrB insertion sites were identified in conditions where the enoA or the alaS genes were overexpressed (small open and black arrowheads) or not (large open and small gray arrowheads) from a P23 constitutive promoter. mRNA chimeras between ltrB-exon 1 (E1) and L. lactis mRNAs (d, E1-enoA)(h, E1-alaS) as well as between L. lactis mRNAs (i, alaS-enoA)(j, enoA-alaS) were also detected by RT-PCR at IBS1/2-like sequences.
Fig 8.
Detection of intermediates unique to the reverse splicing pathway: Ll.LtrB reverse-spliced within an L. lactis mRNA and an E1-mRNA chimera.
RT-PCR assays were performed to detect the 5’ and 3’ junctions of Ll.LtrB-EBS1/Mut-ΔLtrA+LtrA and Ll.LtrB-ΔA-EBS1/Mut-ΔLtrA+LtrA reverse splicing events within the Ribosomal Protein S12/S7 mRNA (a, b). Complete and dashed arrows indicate reverse splicing of Ll.LtrB lariats within strong (S) and weak (W) IBS1/2-like sequences (—|—) respectively. The strong (S)(10/11 nts)(large arrowhead) and weak (W)(8-9/11 nts)(small arrowheads) IBS1/2-like sequences invaded by reverse splicing are represented (c). The sites flanking the mRNA fragment (c, 161 nts) initially detected at intron circle splice junctions (Fig 3B) are indicated by open arrowheads. mRNA chimeras between ltrB-exon 1 (E1) and L. lactis mRNAs (d, E1-S12/S7) were also detected by RT-PCR at IBS1/2-like sequences.
Fig 9.
The intergenic group II intron trans-splicing pathway leading to the generation of alaS-enoA and enoA-alaS mRNA chimeras.
Ll.LtrB first recognizes by base pairing and invades by reverse splicing IBS1/2-like sequences on the enoA (red) and alaS (blue) mRNAs (step 1). The 3’OH of intron-processed alaS mRNA fragments (E1-like alaS fragments), harboring IBS1/2-like sequences (—|—) at their 3’ ends (dark blue and blue)(dark blue), can, similarly to free E1, be recruited by the intron to initiate the circularization pathway. These fragments can attack the intron-exon 2 splice junction of the Ll.LtrB-interrupted enoA mRNA (red) leading to the generation of alaS-enoA mRNA chimeras (step 2). Subsequent excision of the intron by circularization releases enoA mRNA fragments (E1-like enoA fragments) with IBS1/2-like sequences at their 3’ ends (step 3a, dark red and red)(step 3b, dark red), which can in turn initiate the generation of enoA-alaS mRNA chimeras with intron-interrupted alaS mRNA (blue).
Fig 10.
Model for group II intron-catalysed genetic diversity.
Upon expression of group II intron-interrupted genes in bacteria, the ribozymes self-splice using the conventional branching pathway, releasing a mix of RNPs (lariats + LtrA) and accurately ligated flanking exons (step 1). Excised RNPs next interact with cellular mRNA transcripts through specific base pairing with IBS1/2-like sequences (—|—). This interaction leads either to complete reverse splicing or hydrolysis at the IBS1/2-like sites, producing a population of intron-invaded mRNA transcripts or hydrolysed mRNA fragments with a free 3’-OH, respectively (step 2). When introns interrupting an ectopic site self-splice using the circularization pathway, they recruit either processed ectopic mRNA fragments or their processed cognate E1, which can both act as external nucleophiles in an intergenic trans-splicing reaction (step 3). This produces two distinct populations of chimeric mRNA transcripts: E1-mRNA and mRNA-mRNA products, which together increase the overall diversity of the bacterial host’s transcriptome. The presence of a series of group II intron–interrupted mRNAs may potentially lead to a multitude of chimeric mRNA-mRNA combinations.