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A conserved 3' extension in unusual group II introns is important for efficient second-step splicing.

Stabell FB, Tourasse NJ, Kolstø AB - Nucleic Acids Res. (2009)

Bottom Line: Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class.The extension of these introns is predicted to form a conserved two-stem-loop structure.This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3' extension. Here, we report the discovery of four additional group II introns with a similar 3' extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47-61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem-loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.

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In vivo splicing of unusual group II introns in B. thuringiensis kurstaki 4D1. RT-PCR was conducted on total RNA with primers located in the flanking exons. The gel picture shows the RT-PCR products of the spliced exons (names of the products related to each intron are given on top) and the corresponding negative controls run without reverse transcriptase (lanes marked with NC). Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 2.8% NuSieve GTG agarose gel (Cambrex).
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Figure 2: In vivo splicing of unusual group II introns in B. thuringiensis kurstaki 4D1. RT-PCR was conducted on total RNA with primers located in the flanking exons. The gel picture shows the RT-PCR products of the spliced exons (names of the products related to each intron are given on top) and the corresponding negative controls run without reverse transcriptase (lanes marked with NC). Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 2.8% NuSieve GTG agarose gel (Cambrex).

Mentions: Through a sequence similarity search of private sequence collections using BLAST (16) and the 3′ extension of the unusual B.c.I4 intron of B. cereus ATCC 10987 (11,12) as query, four sequence fragments exhibiting similarity to the B.c.I4 extension were identified in B. thuringiensis kurstaki BGSC 4D1 (also known as AH248 or KB004). Further cloning, sequencing and computational secondary structure predictions revealed that all of these sequences contained a full group II intron with the six typical domains and a 3′ extension (Figure 1). No additional group II introns with this unusual extension were identified in a similar search of public sequence databases, as well as in a structural search with RNAMotif (18) (see ‘Material and Methods’ section). Three of the B. thuringiensis kurstaki introns were located in genes homologous to genes from the pXO1 plasmid of B. anthracis (Table 1). These introns were named (according to the nomenclature used in ref. 25) B.th.I5, B.th.I6a and b. The latter two are inserted in different genomic contigs and homologs of a predicted conjugation gene (∼80% nucleotide sequence identity). B.th.I6a and b are 98.4% identical overall and inserted in the same sites thereby representing two copies of the same intron B.th.I6. The remaining intron B.th.I7 was inserted in a homolog of a hypothetical gene from the pBc10987 plasmid of B. cereus ATCC 10987 (Table 1). RT-PCR conducted on total RNA from B. thuringiensis kurstaki 4D1 with host gene-specific primers showed that the B. thuringiensis introns spliced and thus were functional in vivo (Figure 2 and Supplementary Table 1). Sequencing of the RT-PCR products confirmed that the 3′ splice sites of B.th.I5, B.th.I6 and B.th.I7 were located respectively 53, 54 and 54 nucleotides downstream of domain VI, as opposed to the usual three or four bases. In addition, in vitro splicing of B.th.I5 and B.th.I6a confirmed the splice boundaries observed in vivo for these introns (see below). Furthermore, as in the B.c.I4 intron, potential EBS3-IBS3 and γ–γ′ base-pair interaction sites, which are important for 3′ splice site selection (2,26,27) could be identified at the observed 3′ splice site (Figure 1). B.th.I5, B.th.I6 and B.th.I7 therefore represent new examples of bacterial group II introns carrying a 3′ extension. Overall, the four different B. cereus and B. thuringiensis introns are only 47–61% identical at the nucleotide level (31–52% amino acid sequence identity between the ORFs). Phylogenetic analysis of the ORFs of 221 bacterial group II introns available at the group II intron database (17) revealed that these four unusual introns belong to the B class (according to the nomenclature mentioned in ref. 6; Figure 3). However, they do not group in a single lineage but are located in two subgroups herein named α and β. B.c.I4 and B.th.I7 belong to subgroup α, while B.th.I5 and B.th.I6 are in subgroup β. The division of the four introns harboring an extra 3′ segment based on ORF sequence relatedness is supported by the fact that the introns share structural features common to each subgroup (Supplementary Figure 1). These features are all located in domain I of the RNA secondary structure. Despite this divergence, sequence and secondary structure comparisons done both manually and using RNAForester (28,29) revealed that all four introns share several conserved regions (see Supplementary Figures 2 and 3). Besides domains V and VI, which are highly conserved, several nucleotides within the 3′ extra segment are identical (marked in red in Figure 4A). The 3′ extension of the B. thuringiensis introns could fold into two stem–loop structures (S1 and S2) similar to those in B.c.I4 of B. cereus, where the most conserved sites in structure and sequence are within the small stem S1 and the asymmetric internal loop of the longer stem S2. The sequence and folding conservation, together with the occurrence of compensatory mutations in S2, strongly suggest that the 3′ extension forms a stable structure downstream of domain VI for these four unusual introns, and thus might indicate the importance of maintaining this structure for intron function. Remarkably, the S2 internal loop and its surrounding base pairs show a striking resemblance to, and matches the consensus of, the 11-nt tetraloop receptor motif 5′ [CCUAAG … UAUGG] 3′ (30). This is a common RNA motif that participates in the tertiary folding of several catalytic RNAs by interacting with tetraloops of the generic GNn/RA family (30–32). In addition to the 3′ end, there is a high sequence and structure conservation in the stem of subdomain IC1 in B.c.I4, B.th.I5 and B.th.I6, while B.th.I7 shows a lower sequence conservation (see Figures 1 and 4F). Intriguingly, the conserved area is contiguous to the bulged region containing the ϵ′- and λ sites (z-anchor) that form interactions with the 5′ end of the intron and/or domain V (9,33).Figure 4.


A conserved 3' extension in unusual group II introns is important for efficient second-step splicing.

Stabell FB, Tourasse NJ, Kolstø AB - Nucleic Acids Res. (2009)

In vivo splicing of unusual group II introns in B. thuringiensis kurstaki 4D1. RT-PCR was conducted on total RNA with primers located in the flanking exons. The gel picture shows the RT-PCR products of the spliced exons (names of the products related to each intron are given on top) and the corresponding negative controls run without reverse transcriptase (lanes marked with NC). Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 2.8% NuSieve GTG agarose gel (Cambrex).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: In vivo splicing of unusual group II introns in B. thuringiensis kurstaki 4D1. RT-PCR was conducted on total RNA with primers located in the flanking exons. The gel picture shows the RT-PCR products of the spliced exons (names of the products related to each intron are given on top) and the corresponding negative controls run without reverse transcriptase (lanes marked with NC). Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 2.8% NuSieve GTG agarose gel (Cambrex).
Mentions: Through a sequence similarity search of private sequence collections using BLAST (16) and the 3′ extension of the unusual B.c.I4 intron of B. cereus ATCC 10987 (11,12) as query, four sequence fragments exhibiting similarity to the B.c.I4 extension were identified in B. thuringiensis kurstaki BGSC 4D1 (also known as AH248 or KB004). Further cloning, sequencing and computational secondary structure predictions revealed that all of these sequences contained a full group II intron with the six typical domains and a 3′ extension (Figure 1). No additional group II introns with this unusual extension were identified in a similar search of public sequence databases, as well as in a structural search with RNAMotif (18) (see ‘Material and Methods’ section). Three of the B. thuringiensis kurstaki introns were located in genes homologous to genes from the pXO1 plasmid of B. anthracis (Table 1). These introns were named (according to the nomenclature used in ref. 25) B.th.I5, B.th.I6a and b. The latter two are inserted in different genomic contigs and homologs of a predicted conjugation gene (∼80% nucleotide sequence identity). B.th.I6a and b are 98.4% identical overall and inserted in the same sites thereby representing two copies of the same intron B.th.I6. The remaining intron B.th.I7 was inserted in a homolog of a hypothetical gene from the pBc10987 plasmid of B. cereus ATCC 10987 (Table 1). RT-PCR conducted on total RNA from B. thuringiensis kurstaki 4D1 with host gene-specific primers showed that the B. thuringiensis introns spliced and thus were functional in vivo (Figure 2 and Supplementary Table 1). Sequencing of the RT-PCR products confirmed that the 3′ splice sites of B.th.I5, B.th.I6 and B.th.I7 were located respectively 53, 54 and 54 nucleotides downstream of domain VI, as opposed to the usual three or four bases. In addition, in vitro splicing of B.th.I5 and B.th.I6a confirmed the splice boundaries observed in vivo for these introns (see below). Furthermore, as in the B.c.I4 intron, potential EBS3-IBS3 and γ–γ′ base-pair interaction sites, which are important for 3′ splice site selection (2,26,27) could be identified at the observed 3′ splice site (Figure 1). B.th.I5, B.th.I6 and B.th.I7 therefore represent new examples of bacterial group II introns carrying a 3′ extension. Overall, the four different B. cereus and B. thuringiensis introns are only 47–61% identical at the nucleotide level (31–52% amino acid sequence identity between the ORFs). Phylogenetic analysis of the ORFs of 221 bacterial group II introns available at the group II intron database (17) revealed that these four unusual introns belong to the B class (according to the nomenclature mentioned in ref. 6; Figure 3). However, they do not group in a single lineage but are located in two subgroups herein named α and β. B.c.I4 and B.th.I7 belong to subgroup α, while B.th.I5 and B.th.I6 are in subgroup β. The division of the four introns harboring an extra 3′ segment based on ORF sequence relatedness is supported by the fact that the introns share structural features common to each subgroup (Supplementary Figure 1). These features are all located in domain I of the RNA secondary structure. Despite this divergence, sequence and secondary structure comparisons done both manually and using RNAForester (28,29) revealed that all four introns share several conserved regions (see Supplementary Figures 2 and 3). Besides domains V and VI, which are highly conserved, several nucleotides within the 3′ extra segment are identical (marked in red in Figure 4A). The 3′ extension of the B. thuringiensis introns could fold into two stem–loop structures (S1 and S2) similar to those in B.c.I4 of B. cereus, where the most conserved sites in structure and sequence are within the small stem S1 and the asymmetric internal loop of the longer stem S2. The sequence and folding conservation, together with the occurrence of compensatory mutations in S2, strongly suggest that the 3′ extension forms a stable structure downstream of domain VI for these four unusual introns, and thus might indicate the importance of maintaining this structure for intron function. Remarkably, the S2 internal loop and its surrounding base pairs show a striking resemblance to, and matches the consensus of, the 11-nt tetraloop receptor motif 5′ [CCUAAG … UAUGG] 3′ (30). This is a common RNA motif that participates in the tertiary folding of several catalytic RNAs by interacting with tetraloops of the generic GNn/RA family (30–32). In addition to the 3′ end, there is a high sequence and structure conservation in the stem of subdomain IC1 in B.c.I4, B.th.I5 and B.th.I6, while B.th.I7 shows a lower sequence conservation (see Figures 1 and 4F). Intriguingly, the conserved area is contiguous to the bulged region containing the ϵ′- and λ sites (z-anchor) that form interactions with the 5′ end of the intron and/or domain V (9,33).Figure 4.

Bottom Line: Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class.The extension of these introns is predicted to form a conserved two-stem-loop structure.This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3' extension. Here, we report the discovery of four additional group II introns with a similar 3' extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47-61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem-loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.

Show MeSH
Related in: MedlinePlus