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Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA.

Birgisdottir AB, Johansen S - Nucleic Acids Res. (2005)

Bottom Line: The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers.Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site.Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø N-9037 Tromsø, Norway.

ABSTRACT
The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

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Detection of Dir.S956-1 (WT) reverse splicing in S.cerevisiae. (A) RT–PCR amplification on yeast total RNA using primers OP85 and OP1009 resulting in a product of 273 bp. Sequencing of this product confirms the 3′ integration junction of Dir.S956-1 to site 956 of yeast SSU rRNA. (B) Northern blot analysis on 5 μg total RNA isolated from Dir.S956-1 containing yeast cells. The blot with a 32P-UTP labelled HEG riboprobe (indicated in Figure 3A) reveals several intron containing signals: R, reverse spliced product; RI, reverse spliced intron intermediate where the 3′ end of the intron is ligated to the yeast SSU rRNA 3′ exon; I, free intron RNA; PI, DiGIR1 processed free intron. The lower part of the blot assumed to reveal the signal for free intron RNA and intron processing products was hybridized separately. Lanes denoted with a plus indicate that the total RNA is isolated from galactose-induced Dir.S956-1 containing yeast cells and lanes denoted with a minus indicate lack of galactose induction. The positions of LSU (3393 nt) and SSU (1789) yeast rRNAs on the blot as well as the positions of two molecular marker signals (from the 0.28 to 6.58 kb RNA size marker; Promega) are indicated on the left.
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fig4: Detection of Dir.S956-1 (WT) reverse splicing in S.cerevisiae. (A) RT–PCR amplification on yeast total RNA using primers OP85 and OP1009 resulting in a product of 273 bp. Sequencing of this product confirms the 3′ integration junction of Dir.S956-1 to site 956 of yeast SSU rRNA. (B) Northern blot analysis on 5 μg total RNA isolated from Dir.S956-1 containing yeast cells. The blot with a 32P-UTP labelled HEG riboprobe (indicated in Figure 3A) reveals several intron containing signals: R, reverse spliced product; RI, reverse spliced intron intermediate where the 3′ end of the intron is ligated to the yeast SSU rRNA 3′ exon; I, free intron RNA; PI, DiGIR1 processed free intron. The lower part of the blot assumed to reveal the signal for free intron RNA and intron processing products was hybridized separately. Lanes denoted with a plus indicate that the total RNA is isolated from galactose-induced Dir.S956-1 containing yeast cells and lanes denoted with a minus indicate lack of galactose induction. The positions of LSU (3393 nt) and SSU (1789) yeast rRNAs on the blot as well as the positions of two molecular marker signals (from the 0.28 to 6.58 kb RNA size marker; Promega) are indicated on the left.

Mentions: We wanted to test for reverse splicing in eukaryote cells and expressed the Didymium intron in the yeast Saccharomyces cerevisiae. These experiments followed an approach similar to that described for reverse splicing in E.coli (above). The wild-type intron, flanked by 67 bp of 5′ exon and 205 bp of 3′ exon, was inserted into the yeast expression vector pYESM (23) behind the GAL1 promoter. The plasmid denoted pYGAL-Dir.S956-1 was transformed into the S.cerevisiae strain INVSc2. With purified total RNA from galactose-induced yeast cells as template, RT–PCR reactions amplified products consistent with intron integration to site 956 (E.coli SSU rRNA numbering). DNA sequencing of the 3′ intron–exon junction implied integration at site 956 (Figure 4A). In contrast, we were unable to amplify the corresponding 5′ exon–intron integration junction (site 956). The observation that 3′ integration junctions are obtained more readily than 5′ integration junctions has been noted in reverse splicing experiments with the Tetrahymena intron (7,8). Perhaps the first step of reverse splicing is more efficient than the second in yeast. Alternatively, many reverse splicing products resplice in yeast. A northern blot analysis on yeast total RNA, using the HEG-specific probe (Figure 3A), revealed a signal for an RNA of ∼2 kb (Figure 4B) The northern blot analysis was also conducted with the DiGIR2-specific probe giving the same signal pattern (data not shown). The size expected for a reverse splicing intermediate where the 3′ end of Dir.S956-1 is ligated to U957 of the yeast SSU rRNA (the first step of reverse splicing) is 2052 nt. A signal for such an intermediate was not detected in the reverse splicing experiments in E.coli, and indicates a reduced efficiency of the second step of reverse splicing in yeast compared to bacteria. A weak signal (also detected with the DiGIR2 probe) correlating with the size of yeast SSU rRNA harbouring the Didymium intron implies complete integration of Dir.S956-1 into yeast SSU rRNA (Figure 4B). However, due to failure to amplify the 5′ integration junction by RT–PCR, we can only conclude partial reverse splicing in yeast.


Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA.

Birgisdottir AB, Johansen S - Nucleic Acids Res. (2005)

Detection of Dir.S956-1 (WT) reverse splicing in S.cerevisiae. (A) RT–PCR amplification on yeast total RNA using primers OP85 and OP1009 resulting in a product of 273 bp. Sequencing of this product confirms the 3′ integration junction of Dir.S956-1 to site 956 of yeast SSU rRNA. (B) Northern blot analysis on 5 μg total RNA isolated from Dir.S956-1 containing yeast cells. The blot with a 32P-UTP labelled HEG riboprobe (indicated in Figure 3A) reveals several intron containing signals: R, reverse spliced product; RI, reverse spliced intron intermediate where the 3′ end of the intron is ligated to the yeast SSU rRNA 3′ exon; I, free intron RNA; PI, DiGIR1 processed free intron. The lower part of the blot assumed to reveal the signal for free intron RNA and intron processing products was hybridized separately. Lanes denoted with a plus indicate that the total RNA is isolated from galactose-induced Dir.S956-1 containing yeast cells and lanes denoted with a minus indicate lack of galactose induction. The positions of LSU (3393 nt) and SSU (1789) yeast rRNAs on the blot as well as the positions of two molecular marker signals (from the 0.28 to 6.58 kb RNA size marker; Promega) are indicated on the left.
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Related In: Results  -  Collection

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fig4: Detection of Dir.S956-1 (WT) reverse splicing in S.cerevisiae. (A) RT–PCR amplification on yeast total RNA using primers OP85 and OP1009 resulting in a product of 273 bp. Sequencing of this product confirms the 3′ integration junction of Dir.S956-1 to site 956 of yeast SSU rRNA. (B) Northern blot analysis on 5 μg total RNA isolated from Dir.S956-1 containing yeast cells. The blot with a 32P-UTP labelled HEG riboprobe (indicated in Figure 3A) reveals several intron containing signals: R, reverse spliced product; RI, reverse spliced intron intermediate where the 3′ end of the intron is ligated to the yeast SSU rRNA 3′ exon; I, free intron RNA; PI, DiGIR1 processed free intron. The lower part of the blot assumed to reveal the signal for free intron RNA and intron processing products was hybridized separately. Lanes denoted with a plus indicate that the total RNA is isolated from galactose-induced Dir.S956-1 containing yeast cells and lanes denoted with a minus indicate lack of galactose induction. The positions of LSU (3393 nt) and SSU (1789) yeast rRNAs on the blot as well as the positions of two molecular marker signals (from the 0.28 to 6.58 kb RNA size marker; Promega) are indicated on the left.
Mentions: We wanted to test for reverse splicing in eukaryote cells and expressed the Didymium intron in the yeast Saccharomyces cerevisiae. These experiments followed an approach similar to that described for reverse splicing in E.coli (above). The wild-type intron, flanked by 67 bp of 5′ exon and 205 bp of 3′ exon, was inserted into the yeast expression vector pYESM (23) behind the GAL1 promoter. The plasmid denoted pYGAL-Dir.S956-1 was transformed into the S.cerevisiae strain INVSc2. With purified total RNA from galactose-induced yeast cells as template, RT–PCR reactions amplified products consistent with intron integration to site 956 (E.coli SSU rRNA numbering). DNA sequencing of the 3′ intron–exon junction implied integration at site 956 (Figure 4A). In contrast, we were unable to amplify the corresponding 5′ exon–intron integration junction (site 956). The observation that 3′ integration junctions are obtained more readily than 5′ integration junctions has been noted in reverse splicing experiments with the Tetrahymena intron (7,8). Perhaps the first step of reverse splicing is more efficient than the second in yeast. Alternatively, many reverse splicing products resplice in yeast. A northern blot analysis on yeast total RNA, using the HEG-specific probe (Figure 3A), revealed a signal for an RNA of ∼2 kb (Figure 4B) The northern blot analysis was also conducted with the DiGIR2-specific probe giving the same signal pattern (data not shown). The size expected for a reverse splicing intermediate where the 3′ end of Dir.S956-1 is ligated to U957 of the yeast SSU rRNA (the first step of reverse splicing) is 2052 nt. A signal for such an intermediate was not detected in the reverse splicing experiments in E.coli, and indicates a reduced efficiency of the second step of reverse splicing in yeast compared to bacteria. A weak signal (also detected with the DiGIR2 probe) correlating with the size of yeast SSU rRNA harbouring the Didymium intron implies complete integration of Dir.S956-1 into yeast SSU rRNA (Figure 4B). However, due to failure to amplify the 5′ integration junction by RT–PCR, we can only conclude partial reverse splicing in yeast.

Bottom Line: The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers.Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site.Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø N-9037 Tromsø, Norway.

ABSTRACT
The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

Show MeSH
Related in: MedlinePlus