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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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Time-course analysis of in vitro self-splicing of B.c.I4 and B.th.I6a wild-type (WT) and mutant constructs carrying changes in the 3′ extension or in subdomain IC1 (see Figure 4). dS1S2 is a B.c.I4 construct lacking the entire 3′ extension (previously named d56; see ref. 11). Splicing was performed in 40 mM MOPS, pH = 7.5, 100 mM MgCl2 and 500 mM (NH4)2SO4 at 47°C. The relative fractions of unspliced precursor RNA (A, C and E) and released lariat intron (B, D and F) were computed from the intensities of the radioactive bands using a phosphorimager. Reactions were repeated three times for each construct, and are expressed as averages. Data were fitted to a biphasic exponential kinetic model [Equations (6) and (8) in ref. 24, and rate constants are given in Supplementary Table 2].
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Figure 6: Time-course analysis of in vitro self-splicing of B.c.I4 and B.th.I6a wild-type (WT) and mutant constructs carrying changes in the 3′ extension or in subdomain IC1 (see Figure 4). dS1S2 is a B.c.I4 construct lacking the entire 3′ extension (previously named d56; see ref. 11). Splicing was performed in 40 mM MOPS, pH = 7.5, 100 mM MgCl2 and 500 mM (NH4)2SO4 at 47°C. The relative fractions of unspliced precursor RNA (A, C and E) and released lariat intron (B, D and F) were computed from the intensities of the radioactive bands using a phosphorimager. Reactions were repeated three times for each construct, and are expressed as averages. Data were fitted to a biphasic exponential kinetic model [Equations (6) and (8) in ref. 24, and rate constants are given in Supplementary Table 2].

Mentions: Since the extra 3′ element is conserved in structure and partially in sequence between the four B. cereus and B. thuringiensis introns described here, we conducted an in vitro mutational analysis of this element in B.c.I4 in order to investigate whether it contributes to the splicing activity of the intron. In vitro splicing was conducted under the same conditions as in (11), i.e. at 47°C in 0.5 M ammonium sulfate ((NH4)2SO4), 40 mM MOPS, pH = 7.5 and 100 mM MgCl2 (see ‘Material and Methods’ section). First, two deletion mutants were made by removing separately each of the two stem–loop structures of the 3′ extension (S1 and S2) from the B.c.I4 wild-type (WT) ΔORF construct (Figure 4B and C). For the mutant dS2 in which the longer S2 stem was deleted, there was a drastic reduction of the amount of free lariat formed after the second step of splicing, together with a clear increase of the first step intermediate ‘lariat with 3′ exon’, compared to WT intron (Figure 5, two top bands). A time-course kinetic analysis showed that the fraction of unreacted dS2 precursor RNA was ∼20% higher than that of WT, while the relative fraction of lariat released by dS2 was decreased by ∼60% on average (Figure 6A and B). Altogether, these results indicate it is mostly the second splicing reaction that is severely slowed down when the S2 stem–loop structure of the 3′ extension is removed from the intron. To support this argument no clear band corresponding to the ligated exons could be observed for this deletion mutant, even though RT-PCR analysis revealed that it did occur, suggesting that the efficiency of exon ligation was decreased. To determine whether the observed phenomenon applies for other introns carrying the extensions, corresponding deletions of S2 were performed on the B. thuringiensis B.th.I5 and B.th.I6a intron constructs. For both introns, a drastic reduction in the efficiency of the second splicing step was also observed compared to the WT, thus pointing to a general importance of S2 for the splicing of the unusual introns (Figures 5 and 6A and B, and Supplementary Figure 4). In sharp contrast to the dS2 mutant, the B.c.I4 construct in which the smaller S1 stem–loop structure was deleted, dS1, showed a splicing efficiency equal or better than that of the WT construct with respect to both the amount of precursor processed and lariat formed (Figure 6A and B). Furthermore, mutating the sequence of the terminal loop of S1 (mutant mS1, Figure 4C) revealed no negative effect on either of the two splicing steps. Therefore, the smaller S1 stem–loop part of the 3′ extension does not appear to be critical for splicing under the conditions tested in this study.Figure 5.


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)

Time-course analysis of in vitro self-splicing of B.c.I4 and B.th.I6a wild-type (WT) and mutant constructs carrying changes in the 3′ extension or in subdomain IC1 (see Figure 4). dS1S2 is a B.c.I4 construct lacking the entire 3′ extension (previously named d56; see ref. 11). Splicing was performed in 40 mM MOPS, pH = 7.5, 100 mM MgCl2 and 500 mM (NH4)2SO4 at 47°C. The relative fractions of unspliced precursor RNA (A, C and E) and released lariat intron (B, D and F) were computed from the intensities of the radioactive bands using a phosphorimager. Reactions were repeated three times for each construct, and are expressed as averages. Data were fitted to a biphasic exponential kinetic model [Equations (6) and (8) in ref. 24, and rate constants are given in Supplementary Table 2].
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Related In: Results  -  Collection

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Figure 6: Time-course analysis of in vitro self-splicing of B.c.I4 and B.th.I6a wild-type (WT) and mutant constructs carrying changes in the 3′ extension or in subdomain IC1 (see Figure 4). dS1S2 is a B.c.I4 construct lacking the entire 3′ extension (previously named d56; see ref. 11). Splicing was performed in 40 mM MOPS, pH = 7.5, 100 mM MgCl2 and 500 mM (NH4)2SO4 at 47°C. The relative fractions of unspliced precursor RNA (A, C and E) and released lariat intron (B, D and F) were computed from the intensities of the radioactive bands using a phosphorimager. Reactions were repeated three times for each construct, and are expressed as averages. Data were fitted to a biphasic exponential kinetic model [Equations (6) and (8) in ref. 24, and rate constants are given in Supplementary Table 2].
Mentions: Since the extra 3′ element is conserved in structure and partially in sequence between the four B. cereus and B. thuringiensis introns described here, we conducted an in vitro mutational analysis of this element in B.c.I4 in order to investigate whether it contributes to the splicing activity of the intron. In vitro splicing was conducted under the same conditions as in (11), i.e. at 47°C in 0.5 M ammonium sulfate ((NH4)2SO4), 40 mM MOPS, pH = 7.5 and 100 mM MgCl2 (see ‘Material and Methods’ section). First, two deletion mutants were made by removing separately each of the two stem–loop structures of the 3′ extension (S1 and S2) from the B.c.I4 wild-type (WT) ΔORF construct (Figure 4B and C). For the mutant dS2 in which the longer S2 stem was deleted, there was a drastic reduction of the amount of free lariat formed after the second step of splicing, together with a clear increase of the first step intermediate ‘lariat with 3′ exon’, compared to WT intron (Figure 5, two top bands). A time-course kinetic analysis showed that the fraction of unreacted dS2 precursor RNA was ∼20% higher than that of WT, while the relative fraction of lariat released by dS2 was decreased by ∼60% on average (Figure 6A and B). Altogether, these results indicate it is mostly the second splicing reaction that is severely slowed down when the S2 stem–loop structure of the 3′ extension is removed from the intron. To support this argument no clear band corresponding to the ligated exons could be observed for this deletion mutant, even though RT-PCR analysis revealed that it did occur, suggesting that the efficiency of exon ligation was decreased. To determine whether the observed phenomenon applies for other introns carrying the extensions, corresponding deletions of S2 were performed on the B. thuringiensis B.th.I5 and B.th.I6a intron constructs. For both introns, a drastic reduction in the efficiency of the second splicing step was also observed compared to the WT, thus pointing to a general importance of S2 for the splicing of the unusual introns (Figures 5 and 6A and B, and Supplementary Figure 4). In sharp contrast to the dS2 mutant, the B.c.I4 construct in which the smaller S1 stem–loop structure was deleted, dS1, showed a splicing efficiency equal or better than that of the WT construct with respect to both the amount of precursor processed and lariat formed (Figure 6A and B). Furthermore, mutating the sequence of the terminal loop of S1 (mutant mS1, Figure 4C) revealed no negative effect on either of the two splicing steps. Therefore, the smaller S1 stem–loop part of the 3′ extension does not appear to be critical for splicing under the conditions tested in this study.Figure 5.

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