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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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(A) Unrooted phylogenetic tree of 221 bacterial group II intron-encoded proteins. The tree was reconstructed using the maximum-likelihood method (RAxML program) and was based on the RT domains of the proteins. The major intron classes are named as in ref. 21: A-F, ML (mitochondrial-like), CL (chloroplast-like) and UC (unclassified). B-class introns are shaded, with the unusual B. cereus and B. thuringiensis group II introns carrying the 3′ extension indicated by name and a black square. Unlike in the tree as in ref. 21, introns from the CL2A and CL2B subclasses are grouped together in the present tree. (B) Detailed rooted phylogenetic tree of the B class group II introns, built the same way as in (A), but based on amino acid sequences covering the full length of the intron encoded ORF. The unusual introns are shown in bold and indicated with asterisks. Information, sequences and secondary structure models of all other introns can be found in the Group II intron database [http://www.fp.ucalgary.ca/group2introns/; (17)]. Species names are abbreviated as follows: Ba.sp, Bacillus sp.; B.a, Bacillus anthracis; B.c, Bacillus cereus, B.me, Bacillus megaterium, B.th, Bacillus thuringiensis, C.d, Clostridium difficile, Cl.pe, Clostridium perfringens, E.f, Enterococcus fæcalis, En.fm, Enterococcus fæcium; and G.k, Geobacillus kaustophilus. In (A) and (B) numbers next to branch nodes indicate bootstrap support values (in percentage out of 1000 replicates). Scale bars are in average numbers of amino acid substitutions per site. Proposed subgroupings within the B class are labeled α and β.
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Figure 3: (A) Unrooted phylogenetic tree of 221 bacterial group II intron-encoded proteins. The tree was reconstructed using the maximum-likelihood method (RAxML program) and was based on the RT domains of the proteins. The major intron classes are named as in ref. 21: A-F, ML (mitochondrial-like), CL (chloroplast-like) and UC (unclassified). B-class introns are shaded, with the unusual B. cereus and B. thuringiensis group II introns carrying the 3′ extension indicated by name and a black square. Unlike in the tree as in ref. 21, introns from the CL2A and CL2B subclasses are grouped together in the present tree. (B) Detailed rooted phylogenetic tree of the B class group II introns, built the same way as in (A), but based on amino acid sequences covering the full length of the intron encoded ORF. The unusual introns are shown in bold and indicated with asterisks. Information, sequences and secondary structure models of all other introns can be found in the Group II intron database [http://www.fp.ucalgary.ca/group2introns/; (17)]. Species names are abbreviated as follows: Ba.sp, Bacillus sp.; B.a, Bacillus anthracis; B.c, Bacillus cereus, B.me, Bacillus megaterium, B.th, Bacillus thuringiensis, C.d, Clostridium difficile, Cl.pe, Clostridium perfringens, E.f, Enterococcus fæcalis, En.fm, Enterococcus fæcium; and G.k, Geobacillus kaustophilus. In (A) and (B) numbers next to branch nodes indicate bootstrap support values (in percentage out of 1000 replicates). Scale bars are in average numbers of amino acid substitutions per site. Proposed subgroupings within the B class are labeled α and β.

Mentions: Group II introns are self-splicing ribozymes that are able to excise themselves from precursor mRNA transcripts. They are also retroelements which encode a multifunctional reverse-transcriptase (RT) open reading frame (ORF) and through reverse-splicing they are able to invade new DNA locations (1–5). They are found in the genomes of bacteria, archaea and eukaryotic organelles. Phylogenetically, group II introns can be divided into several major subfamilies based on RNA secondary structure features and ORF sequences [Figure 3A; (1,5–7)]. The secondary structure of group II intron RNA consists of six domains that are linked by a network of tertiary interactions (2,8–10). In particular, domain I forms the scaffold for intron assembly and domain V is essential for catalysis. The other structural elements are important for compaction, stabilization and/or catalysis. Group II intron splicing proceeds through two transesterification reactions. The first reaction is mediated via nucleophilic attack on the 5′ intron–exon junction either by the 2′ hydroxyl group of the bulged adenosine in domain VI or by water. Subsequently the flanking exons are ligated and a branched intron lariat or a linear intron form is respectively released (1–3).Figure 1.


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)

(A) Unrooted phylogenetic tree of 221 bacterial group II intron-encoded proteins. The tree was reconstructed using the maximum-likelihood method (RAxML program) and was based on the RT domains of the proteins. The major intron classes are named as in ref. 21: A-F, ML (mitochondrial-like), CL (chloroplast-like) and UC (unclassified). B-class introns are shaded, with the unusual B. cereus and B. thuringiensis group II introns carrying the 3′ extension indicated by name and a black square. Unlike in the tree as in ref. 21, introns from the CL2A and CL2B subclasses are grouped together in the present tree. (B) Detailed rooted phylogenetic tree of the B class group II introns, built the same way as in (A), but based on amino acid sequences covering the full length of the intron encoded ORF. The unusual introns are shown in bold and indicated with asterisks. Information, sequences and secondary structure models of all other introns can be found in the Group II intron database [http://www.fp.ucalgary.ca/group2introns/; (17)]. Species names are abbreviated as follows: Ba.sp, Bacillus sp.; B.a, Bacillus anthracis; B.c, Bacillus cereus, B.me, Bacillus megaterium, B.th, Bacillus thuringiensis, C.d, Clostridium difficile, Cl.pe, Clostridium perfringens, E.f, Enterococcus fæcalis, En.fm, Enterococcus fæcium; and G.k, Geobacillus kaustophilus. In (A) and (B) numbers next to branch nodes indicate bootstrap support values (in percentage out of 1000 replicates). Scale bars are in average numbers of amino acid substitutions per site. Proposed subgroupings within the B class are labeled α and β.
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Related In: Results  -  Collection

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Figure 3: (A) Unrooted phylogenetic tree of 221 bacterial group II intron-encoded proteins. The tree was reconstructed using the maximum-likelihood method (RAxML program) and was based on the RT domains of the proteins. The major intron classes are named as in ref. 21: A-F, ML (mitochondrial-like), CL (chloroplast-like) and UC (unclassified). B-class introns are shaded, with the unusual B. cereus and B. thuringiensis group II introns carrying the 3′ extension indicated by name and a black square. Unlike in the tree as in ref. 21, introns from the CL2A and CL2B subclasses are grouped together in the present tree. (B) Detailed rooted phylogenetic tree of the B class group II introns, built the same way as in (A), but based on amino acid sequences covering the full length of the intron encoded ORF. The unusual introns are shown in bold and indicated with asterisks. Information, sequences and secondary structure models of all other introns can be found in the Group II intron database [http://www.fp.ucalgary.ca/group2introns/; (17)]. Species names are abbreviated as follows: Ba.sp, Bacillus sp.; B.a, Bacillus anthracis; B.c, Bacillus cereus, B.me, Bacillus megaterium, B.th, Bacillus thuringiensis, C.d, Clostridium difficile, Cl.pe, Clostridium perfringens, E.f, Enterococcus fæcalis, En.fm, Enterococcus fæcium; and G.k, Geobacillus kaustophilus. In (A) and (B) numbers next to branch nodes indicate bootstrap support values (in percentage out of 1000 replicates). Scale bars are in average numbers of amino acid substitutions per site. Proposed subgroupings within the B class are labeled α and β.
Mentions: Group II introns are self-splicing ribozymes that are able to excise themselves from precursor mRNA transcripts. They are also retroelements which encode a multifunctional reverse-transcriptase (RT) open reading frame (ORF) and through reverse-splicing they are able to invade new DNA locations (1–5). They are found in the genomes of bacteria, archaea and eukaryotic organelles. Phylogenetically, group II introns can be divided into several major subfamilies based on RNA secondary structure features and ORF sequences [Figure 3A; (1,5–7)]. The secondary structure of group II intron RNA consists of six domains that are linked by a network of tertiary interactions (2,8–10). In particular, domain I forms the scaffold for intron assembly and domain V is essential for catalysis. The other structural elements are important for compaction, stabilization and/or catalysis. Group II intron splicing proceeds through two transesterification reactions. The first reaction is mediated via nucleophilic attack on the 5′ intron–exon junction either by the 2′ hydroxyl group of the bulged adenosine in domain VI or by water. Subsequently the flanking exons are ligated and a branched intron lariat or a linear intron form is respectively released (1–3).Figure 1.

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