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Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

View Article: PubMed Central - PubMed

ABSTRACT

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

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Related in: MedlinePlus

Reconstitution of RNP particles with the MBP-FlagIEP fusion protein and in vitro-synthesized intron RNA. (A) Lariat formation during the RmInt1 RNP particles reconstitution process was estimated by primer extension. RNP particles reconstituted with different ratios of ΔORF RNA precursor and MBP-FlagIEP fusion protein were subjected to phenol extraction and used as the template for reverse transcription reactions with an intron-specific primer. The intron lariat gave rise to a 97 nt cDNA, whereas the RNA precursor generated a 112 nt product. In vitro-transcribed RNAs corresponding to the precursor and lariat RNA molecules were used as controls. The percentage lariat formation is plotted below. Results were verified by analyses of at least three different IEP preparations and are presented as the mean ± standard error. (B) DNA endonuclease activity on 32P-labeled single stranded DNA substrates (70 nt). DNA cleavage activity resulted in a 35 nt product. The control samples are shown in the left panel: neither RNA nor MBP-FlagIEP, only RNA precursor, only MBP-FlagIEP, and RNA precursor with a domain V mutation (*) plus MBP-FlagIEP. In the middle panel, the DNA substrate was incubated with RNP particles reconstituted from a constant amount of RNA precursor (2 pmol) and increasing amounts of MBP-FlagIEP (10, 25, and 50 pmol). In the right panel, the DNA substrate was incubated with RNP particles reconstituted from a fixed amount of MBP-FlagIEP (100 pmol) and increasing amounts of ΔORF RNA precursor incubated with the ssDNA target substrate. (C) Cleavage efficiency for 70 nt single-stranded DNA substrates 32P-labeled at the 5′ and 3′ ends, with RNP particles reconstituted from 5 μM wild-type to mutant IEPs and 2.5 μM ΔORF precursor RNA, determined after 2 h of incubation. The molecules resulting from the DNA endonuclease reaction are indicated on the right (not drawn to scale): The white box represents the 5′ exon and the black rectangle corresponds to the 3′ exon; the black line identifies the intron RNA.
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Figure 4: Reconstitution of RNP particles with the MBP-FlagIEP fusion protein and in vitro-synthesized intron RNA. (A) Lariat formation during the RmInt1 RNP particles reconstitution process was estimated by primer extension. RNP particles reconstituted with different ratios of ΔORF RNA precursor and MBP-FlagIEP fusion protein were subjected to phenol extraction and used as the template for reverse transcription reactions with an intron-specific primer. The intron lariat gave rise to a 97 nt cDNA, whereas the RNA precursor generated a 112 nt product. In vitro-transcribed RNAs corresponding to the precursor and lariat RNA molecules were used as controls. The percentage lariat formation is plotted below. Results were verified by analyses of at least three different IEP preparations and are presented as the mean ± standard error. (B) DNA endonuclease activity on 32P-labeled single stranded DNA substrates (70 nt). DNA cleavage activity resulted in a 35 nt product. The control samples are shown in the left panel: neither RNA nor MBP-FlagIEP, only RNA precursor, only MBP-FlagIEP, and RNA precursor with a domain V mutation (*) plus MBP-FlagIEP. In the middle panel, the DNA substrate was incubated with RNP particles reconstituted from a constant amount of RNA precursor (2 pmol) and increasing amounts of MBP-FlagIEP (10, 25, and 50 pmol). In the right panel, the DNA substrate was incubated with RNP particles reconstituted from a fixed amount of MBP-FlagIEP (100 pmol) and increasing amounts of ΔORF RNA precursor incubated with the ssDNA target substrate. (C) Cleavage efficiency for 70 nt single-stranded DNA substrates 32P-labeled at the 5′ and 3′ ends, with RNP particles reconstituted from 5 μM wild-type to mutant IEPs and 2.5 μM ΔORF precursor RNA, determined after 2 h of incubation. The molecules resulting from the DNA endonuclease reaction are indicated on the right (not drawn to scale): The white box represents the 5′ exon and the black rectangle corresponds to the 3′ exon; the black line identifies the intron RNA.

Mentions: We investigated whether the IEP and the excised lariat RNA were sufficient for RmInt1 DNA endonuclease activity, by reconstituting RNP particles from the components purified in vitro, as previously described by Saldanha et al. (1999). The ΔORF precursor RNA was incubated with MBP-FlagIEP, in different ratios, at 30°C for 30–60 min. These experiments assessed the ability of the protein to promote RmInt1 RNA splicing, assuming that the complex remained intact to generate active RNP particles containing the protein and the intron lariat. We tested this hypothesis, by evaluating lariat formation by primer extension (Figure 4A). The cDNA was synthesized with an intron-specific primer complementary to a sequence located 80–97 nucleotides from the 5′ end of the intron (Muñoz-Adelantado et al., 2003). The unspliced precursor-derived products were detected as a 112 nt band, whereas the cDNA corresponding to the lariat molecules migrated at 97 nt. As expected, the amount of lariat formed increased with the amount of MBP-FlagIEP added, before reaching a plateau at about 30% lariat (ratio RNA:IEP 1:50), with no further increase even if the amount of protein was doubled. This finding is consistent with those for our IEP-assisted splicing experiments (Figure 3), suggesting that considerable large proportion of intron RNA cannot take part in the reaction.


Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles
Reconstitution of RNP particles with the MBP-FlagIEP fusion protein and in vitro-synthesized intron RNA. (A) Lariat formation during the RmInt1 RNP particles reconstitution process was estimated by primer extension. RNP particles reconstituted with different ratios of ΔORF RNA precursor and MBP-FlagIEP fusion protein were subjected to phenol extraction and used as the template for reverse transcription reactions with an intron-specific primer. The intron lariat gave rise to a 97 nt cDNA, whereas the RNA precursor generated a 112 nt product. In vitro-transcribed RNAs corresponding to the precursor and lariat RNA molecules were used as controls. The percentage lariat formation is plotted below. Results were verified by analyses of at least three different IEP preparations and are presented as the mean ± standard error. (B) DNA endonuclease activity on 32P-labeled single stranded DNA substrates (70 nt). DNA cleavage activity resulted in a 35 nt product. The control samples are shown in the left panel: neither RNA nor MBP-FlagIEP, only RNA precursor, only MBP-FlagIEP, and RNA precursor with a domain V mutation (*) plus MBP-FlagIEP. In the middle panel, the DNA substrate was incubated with RNP particles reconstituted from a constant amount of RNA precursor (2 pmol) and increasing amounts of MBP-FlagIEP (10, 25, and 50 pmol). In the right panel, the DNA substrate was incubated with RNP particles reconstituted from a fixed amount of MBP-FlagIEP (100 pmol) and increasing amounts of ΔORF RNA precursor incubated with the ssDNA target substrate. (C) Cleavage efficiency for 70 nt single-stranded DNA substrates 32P-labeled at the 5′ and 3′ ends, with RNP particles reconstituted from 5 μM wild-type to mutant IEPs and 2.5 μM ΔORF precursor RNA, determined after 2 h of incubation. The molecules resulting from the DNA endonuclease reaction are indicated on the right (not drawn to scale): The white box represents the 5′ exon and the black rectangle corresponds to the 3′ exon; the black line identifies the intron RNA.
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Figure 4: Reconstitution of RNP particles with the MBP-FlagIEP fusion protein and in vitro-synthesized intron RNA. (A) Lariat formation during the RmInt1 RNP particles reconstitution process was estimated by primer extension. RNP particles reconstituted with different ratios of ΔORF RNA precursor and MBP-FlagIEP fusion protein were subjected to phenol extraction and used as the template for reverse transcription reactions with an intron-specific primer. The intron lariat gave rise to a 97 nt cDNA, whereas the RNA precursor generated a 112 nt product. In vitro-transcribed RNAs corresponding to the precursor and lariat RNA molecules were used as controls. The percentage lariat formation is plotted below. Results were verified by analyses of at least three different IEP preparations and are presented as the mean ± standard error. (B) DNA endonuclease activity on 32P-labeled single stranded DNA substrates (70 nt). DNA cleavage activity resulted in a 35 nt product. The control samples are shown in the left panel: neither RNA nor MBP-FlagIEP, only RNA precursor, only MBP-FlagIEP, and RNA precursor with a domain V mutation (*) plus MBP-FlagIEP. In the middle panel, the DNA substrate was incubated with RNP particles reconstituted from a constant amount of RNA precursor (2 pmol) and increasing amounts of MBP-FlagIEP (10, 25, and 50 pmol). In the right panel, the DNA substrate was incubated with RNP particles reconstituted from a fixed amount of MBP-FlagIEP (100 pmol) and increasing amounts of ΔORF RNA precursor incubated with the ssDNA target substrate. (C) Cleavage efficiency for 70 nt single-stranded DNA substrates 32P-labeled at the 5′ and 3′ ends, with RNP particles reconstituted from 5 μM wild-type to mutant IEPs and 2.5 μM ΔORF precursor RNA, determined after 2 h of incubation. The molecules resulting from the DNA endonuclease reaction are indicated on the right (not drawn to scale): The white box represents the 5′ exon and the black rectangle corresponds to the 3′ exon; the black line identifies the intron RNA.
Mentions: We investigated whether the IEP and the excised lariat RNA were sufficient for RmInt1 DNA endonuclease activity, by reconstituting RNP particles from the components purified in vitro, as previously described by Saldanha et al. (1999). The ΔORF precursor RNA was incubated with MBP-FlagIEP, in different ratios, at 30°C for 30–60 min. These experiments assessed the ability of the protein to promote RmInt1 RNA splicing, assuming that the complex remained intact to generate active RNP particles containing the protein and the intron lariat. We tested this hypothesis, by evaluating lariat formation by primer extension (Figure 4A). The cDNA was synthesized with an intron-specific primer complementary to a sequence located 80–97 nucleotides from the 5′ end of the intron (Muñoz-Adelantado et al., 2003). The unspliced precursor-derived products were detected as a 112 nt band, whereas the cDNA corresponding to the lariat molecules migrated at 97 nt. As expected, the amount of lariat formed increased with the amount of MBP-FlagIEP added, before reaching a plateau at about 30% lariat (ratio RNA:IEP 1:50), with no further increase even if the amount of protein was doubled. This finding is consistent with those for our IEP-assisted splicing experiments (Figure 3), suggesting that considerable large proportion of intron RNA cannot take part in the reaction.

View Article: PubMed Central - PubMed

ABSTRACT

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

No MeSH data available.


Related in: MedlinePlus