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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 in vitro integration of DiGIR2 (EC) circular or linear intron RNAs into an E.coli rRNA substrate by RT–PCR and sequencing: 5 pmol PAGE-purified linear or circular DiGIR2 (EC) and 5 pmol PAGE-purified substrate RNA containing S956 were incubated under reverse splicing conditions [40 mM Tris–HCl (pH 7.5), 50 mM MgCl2, 200 mM KCl, 2 mM spermidine, 5 mM DTT, 50°C]. After 120 min incubation time, products for 5′ and 3′ intron integration junctions (318 and 238 bp, respectively) were amplified by RT–PCR for both linear and circular DiGIR2 RNAs. The RT–PCR products presented here are from the experiment with the linear intron, but a similar band pattern was obtained for the intron circle. The primer pairs used for amplification of intron integration junctions were OP621 and OP236 for the 5′ junction, and OP622 and OP85 for the 3′ junction. The RNA sequences flanking the observed integration junctions (denoted with a diamond) at site 956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. The product of 289 bp represents partial reverse splicing to site 905 in the rRNA. Other visible RT–PCR products are caused by non-specific primer annealing during the RT reaction. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
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fig5: Detection of in vitro integration of DiGIR2 (EC) circular or linear intron RNAs into an E.coli rRNA substrate by RT–PCR and sequencing: 5 pmol PAGE-purified linear or circular DiGIR2 (EC) and 5 pmol PAGE-purified substrate RNA containing S956 were incubated under reverse splicing conditions [40 mM Tris–HCl (pH 7.5), 50 mM MgCl2, 200 mM KCl, 2 mM spermidine, 5 mM DTT, 50°C]. After 120 min incubation time, products for 5′ and 3′ intron integration junctions (318 and 238 bp, respectively) were amplified by RT–PCR for both linear and circular DiGIR2 RNAs. The RT–PCR products presented here are from the experiment with the linear intron, but a similar band pattern was obtained for the intron circle. The primer pairs used for amplification of intron integration junctions were OP621 and OP236 for the 5′ junction, and OP622 and OP85 for the 3′ junction. The RNA sequences flanking the observed integration junctions (denoted with a diamond) at site 956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. The product of 289 bp represents partial reverse splicing to site 905 in the rRNA. Other visible RT–PCR products are caused by non-specific primer annealing during the RT reaction. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).

Mentions: It has previously been speculated that full-length intron RNA circles may have a role in intron horizontal transfers (13,25). The DiGIR2 ribozyme catalyses the formation of RNA circles that contain the entire intron sequence (12,13). We investigated the potential for these circles to integrate in vitro into a substrate derived from the E.coli SSU rRNA (positions 800–1115). For simplicity, we used the minimal DiGIR2 intron construct (i.e. DiGIR1 and HEG deleted, see Figure 3A) that efficiently catalyses intron full-length circle formation as well as reverse splicing (above). As a control, we tested in parallel in vitro reverse splicing of linear DiGIR2 intron RNAs. Equal amounts (5 pmol) of PAGE-purified substrate RNA (part of E.coli SSU rRNA containing S956), and PAGE-purified circular or linear DiGIR2 (EC) RNAs (with the IGS specific for S956) were incubated under conditions that favour reverse splicing [see (5); Materials and Methods]. After 120 min of incubation, the RNAs were used as templates in RT–PCR reactions as described above. Products for 5′ and 3′ integration junctions (318 and 238 bp, respectively) were amplified for both the circular and linear intron RNAs (Figure 5), and subsequent sequencing analysis implied intron integration after nucleotide U956 in the E.coli SSU rRNA transcript (Figure 5). An additional RT–PCR product of 289 bp (Figure 5) indicated partial intron integration after position 905 in the SSU rRNA substrate (i.e. only the 3′ intron–exon junction was found). To summarize, these results suggest that DiGIR2 (EC) circles as well as the linear form of intron are able to integrate in vitro into E.coli SSU rRNA substrate.


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

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

Detection of in vitro integration of DiGIR2 (EC) circular or linear intron RNAs into an E.coli rRNA substrate by RT–PCR and sequencing: 5 pmol PAGE-purified linear or circular DiGIR2 (EC) and 5 pmol PAGE-purified substrate RNA containing S956 were incubated under reverse splicing conditions [40 mM Tris–HCl (pH 7.5), 50 mM MgCl2, 200 mM KCl, 2 mM spermidine, 5 mM DTT, 50°C]. After 120 min incubation time, products for 5′ and 3′ intron integration junctions (318 and 238 bp, respectively) were amplified by RT–PCR for both linear and circular DiGIR2 RNAs. The RT–PCR products presented here are from the experiment with the linear intron, but a similar band pattern was obtained for the intron circle. The primer pairs used for amplification of intron integration junctions were OP621 and OP236 for the 5′ junction, and OP622 and OP85 for the 3′ junction. The RNA sequences flanking the observed integration junctions (denoted with a diamond) at site 956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. The product of 289 bp represents partial reverse splicing to site 905 in the rRNA. Other visible RT–PCR products are caused by non-specific primer annealing during the RT reaction. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074745&req=5

fig5: Detection of in vitro integration of DiGIR2 (EC) circular or linear intron RNAs into an E.coli rRNA substrate by RT–PCR and sequencing: 5 pmol PAGE-purified linear or circular DiGIR2 (EC) and 5 pmol PAGE-purified substrate RNA containing S956 were incubated under reverse splicing conditions [40 mM Tris–HCl (pH 7.5), 50 mM MgCl2, 200 mM KCl, 2 mM spermidine, 5 mM DTT, 50°C]. After 120 min incubation time, products for 5′ and 3′ intron integration junctions (318 and 238 bp, respectively) were amplified by RT–PCR for both linear and circular DiGIR2 RNAs. The RT–PCR products presented here are from the experiment with the linear intron, but a similar band pattern was obtained for the intron circle. The primer pairs used for amplification of intron integration junctions were OP621 and OP236 for the 5′ junction, and OP622 and OP85 for the 3′ junction. The RNA sequences flanking the observed integration junctions (denoted with a diamond) at site 956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. The product of 289 bp represents partial reverse splicing to site 905 in the rRNA. Other visible RT–PCR products are caused by non-specific primer annealing during the RT reaction. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
Mentions: It has previously been speculated that full-length intron RNA circles may have a role in intron horizontal transfers (13,25). The DiGIR2 ribozyme catalyses the formation of RNA circles that contain the entire intron sequence (12,13). We investigated the potential for these circles to integrate in vitro into a substrate derived from the E.coli SSU rRNA (positions 800–1115). For simplicity, we used the minimal DiGIR2 intron construct (i.e. DiGIR1 and HEG deleted, see Figure 3A) that efficiently catalyses intron full-length circle formation as well as reverse splicing (above). As a control, we tested in parallel in vitro reverse splicing of linear DiGIR2 intron RNAs. Equal amounts (5 pmol) of PAGE-purified substrate RNA (part of E.coli SSU rRNA containing S956), and PAGE-purified circular or linear DiGIR2 (EC) RNAs (with the IGS specific for S956) were incubated under conditions that favour reverse splicing [see (5); Materials and Methods]. After 120 min of incubation, the RNAs were used as templates in RT–PCR reactions as described above. Products for 5′ and 3′ integration junctions (318 and 238 bp, respectively) were amplified for both the circular and linear intron RNAs (Figure 5), and subsequent sequencing analysis implied intron integration after nucleotide U956 in the E.coli SSU rRNA transcript (Figure 5). An additional RT–PCR product of 289 bp (Figure 5) indicated partial intron integration after position 905 in the SSU rRNA substrate (i.e. only the 3′ intron–exon junction was found). To summarize, these results suggest that DiGIR2 (EC) circles as well as the linear form of intron are able to integrate in vitro into E.coli SSU rRNA substrate.

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