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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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Reverse splicing of Dir.S956-1 (EC) in E.coli. (A) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli. An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible Ptac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. (B) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. (C) RT–PCR amplification and sequence analysis of intron–E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
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fig2: Reverse splicing of Dir.S956-1 (EC) in E.coli. (A) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli. An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible Ptac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. (B) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. (C) RT–PCR amplification and sequence analysis of intron–E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).

Mentions: We investigated the ability of the mobile Dir.S956-1 twin-ribozyme intron to reverse splice in E.coli. Dir.S956-1 was targeted against position 956 in the bacterial SSU rRNA (S956). This position is homologous to the natural intron insertion site in Didymium even though the sequences surrounding the sites are not identical (5′-GUGGUU956UAAUUC in E.coli and 5′-GCGGCU956UAAUUU in Didymium). During the first step of reverse splicing, a stretch of usually 4–6 nt upstream of the integration site is recognized by the intron internal guide sequence (IGS) (5). The IGS of Dir.S956-1 was therefore changed from 5′-GGCCGC to 5′-GACCAC (Figure 1) to allow correct base pairing between the IGS and the E.coli SSU target sequence. This intron construct was designated Dir.S956-1 (EC). The highly expressed endogenous SSU rRNA provides an abundant substrate for reverse splicing. Dir.S956-1 (EC) with short flanking exon sequences (11 nt of 5′ exon and 10 nt of 3′ exon) was expressed in bacteria from the pMAL-c2X expression vector, under the control of the IPTG-inducible Ptac promoter (Figure 2A). Forward intron splicing from the vector transcripts is a prerequisite for reverse splicing in E.coli. Two hours after IPTG-induction, total RNA was isolated from intron-containing cells and the formation of free intron RNAs in E.coli was indirectly monitored by RT–PCR amplification of ligated exon sequences (data not shown). In addition, free intron RNAs were directly visualized by northern blot analyses (see below). After verifying successful intron splicing, we searched for reverse splicing products. DNase-treated isolated total RNA was subjected to the RT–PCR approach presented in Figure 2B. Different primer pairs (i.e. one intron-specific and one SSU rDNA-specific) were used to amplify across 5′and 3′ intron–exon junctions in the E.coli SSU rRNA (see Materials and Methods for details). We expected the intron to potentially target SSU rRNA sequences with only limited match to the intron IGS sequence, and therefore designed primers that would cover the entire E.coli SSU rRNA. A number of distinct RT–PCR products, amplified with the different primer pairs, were cut from gels, purified and directly DNA sequenced. However, intron integration was detected at S956 exclusively. Sequencing of the RT–PCR products of 238 and 390 bp, from the primer pairs covering S956, revealed 3′ and 5′ intron integration junctions, respectively to site 956 (Figure 2C). The other analysed RT–PCR products also revealed integration at S956 or were caused by non-specific primer annealing (data not shown). PCR reactions, with the respective primer pairs covering site 956, on the purified total RNA (without the RT reaction) and on isolated total DNA from the same bacteria did not amplify the products indicative of intron integration at site 956 (data not shown). Thus, we can conclude that the amplified RT–PCR products arise from RNA and that the integration event we observe is limited to reverse intron splicing at the RNA level. In summary, our results show that Dir.S956-1 intron RNAs reverse splice into site 956 in E.coli SSU rRNA. This position is homologous to the natural splice junction in Didymium.


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

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

Reverse splicing of Dir.S956-1 (EC) in E.coli. (A) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli. An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible Ptac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. (B) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. (C) RT–PCR amplification and sequence analysis of intron–E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
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Related In: Results  -  Collection

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fig2: Reverse splicing of Dir.S956-1 (EC) in E.coli. (A) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli. An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible Ptac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. (B) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. (C) RT–PCR amplification and sequence analysis of intron–E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
Mentions: We investigated the ability of the mobile Dir.S956-1 twin-ribozyme intron to reverse splice in E.coli. Dir.S956-1 was targeted against position 956 in the bacterial SSU rRNA (S956). This position is homologous to the natural intron insertion site in Didymium even though the sequences surrounding the sites are not identical (5′-GUGGUU956UAAUUC in E.coli and 5′-GCGGCU956UAAUUU in Didymium). During the first step of reverse splicing, a stretch of usually 4–6 nt upstream of the integration site is recognized by the intron internal guide sequence (IGS) (5). The IGS of Dir.S956-1 was therefore changed from 5′-GGCCGC to 5′-GACCAC (Figure 1) to allow correct base pairing between the IGS and the E.coli SSU target sequence. This intron construct was designated Dir.S956-1 (EC). The highly expressed endogenous SSU rRNA provides an abundant substrate for reverse splicing. Dir.S956-1 (EC) with short flanking exon sequences (11 nt of 5′ exon and 10 nt of 3′ exon) was expressed in bacteria from the pMAL-c2X expression vector, under the control of the IPTG-inducible Ptac promoter (Figure 2A). Forward intron splicing from the vector transcripts is a prerequisite for reverse splicing in E.coli. Two hours after IPTG-induction, total RNA was isolated from intron-containing cells and the formation of free intron RNAs in E.coli was indirectly monitored by RT–PCR amplification of ligated exon sequences (data not shown). In addition, free intron RNAs were directly visualized by northern blot analyses (see below). After verifying successful intron splicing, we searched for reverse splicing products. DNase-treated isolated total RNA was subjected to the RT–PCR approach presented in Figure 2B. Different primer pairs (i.e. one intron-specific and one SSU rDNA-specific) were used to amplify across 5′and 3′ intron–exon junctions in the E.coli SSU rRNA (see Materials and Methods for details). We expected the intron to potentially target SSU rRNA sequences with only limited match to the intron IGS sequence, and therefore designed primers that would cover the entire E.coli SSU rRNA. A number of distinct RT–PCR products, amplified with the different primer pairs, were cut from gels, purified and directly DNA sequenced. However, intron integration was detected at S956 exclusively. Sequencing of the RT–PCR products of 238 and 390 bp, from the primer pairs covering S956, revealed 3′ and 5′ intron integration junctions, respectively to site 956 (Figure 2C). The other analysed RT–PCR products also revealed integration at S956 or were caused by non-specific primer annealing (data not shown). PCR reactions, with the respective primer pairs covering site 956, on the purified total RNA (without the RT reaction) and on isolated total DNA from the same bacteria did not amplify the products indicative of intron integration at site 956 (data not shown). Thus, we can conclude that the amplified RT–PCR products arise from RNA and that the integration event we observe is limited to reverse intron splicing at the RNA level. In summary, our results show that Dir.S956-1 intron RNAs reverse splice into site 956 in E.coli SSU rRNA. This position is homologous to the natural splice junction in Didymium.

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