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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.

No MeSH data available.


Related in: MedlinePlus

RmInt1 splicing efficiency in vitro. (A) The left panel (lanes 1–2) shows the inability of RmInt1 to self-splice in vitro. Splicing reactions were carried out by incubating 32P-labeled, 905 nt RmInt1 ΔORF RNA with 5 mM MgCl2 for 0 and 300 min. The panels on the right (lanes 3–18) show RmInt1 IEP-assisted splicing, as evaluated by incubating a 32P-labeled ΔORF RNA precursor with different protein preparations in a buffer containing 5 mM MgCl2 for various times. The radioactively labeled precursor ΔORF RNA was incubated with a 50-fold excess of wild- type MBP-FlagIEP fusion protein (middle panel) or RmInt1 IEP mutant proteins (right panel) for 0, 5, 30, and 120 min. The arrows on the right indicate the products obtained: La, fully spliced lariat RNA; and, Pre, precursor RmInt1 ΔORF RNA. (B) Kinetic data, including additional time points, are plotted as the percentage lariat formation relative to the level of precursor RNA. Lines in black/squares represent the MBP-FlagIEP wild-type fusion protein; the RT-deficient mutant (MBP-FlagYAHH) is indicated by a broken black line/triangles; maturase domain mutants are indicated with a solid gray line/circles (MBP-FlagA354A355) or a broken line/diamonds (MBP-FlagA381). The experiment was repeated twice, with at least two independent purified fusion proteins for each mutant.
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Figure 3: RmInt1 splicing efficiency in vitro. (A) The left panel (lanes 1–2) shows the inability of RmInt1 to self-splice in vitro. Splicing reactions were carried out by incubating 32P-labeled, 905 nt RmInt1 ΔORF RNA with 5 mM MgCl2 for 0 and 300 min. The panels on the right (lanes 3–18) show RmInt1 IEP-assisted splicing, as evaluated by incubating a 32P-labeled ΔORF RNA precursor with different protein preparations in a buffer containing 5 mM MgCl2 for various times. The radioactively labeled precursor ΔORF RNA was incubated with a 50-fold excess of wild- type MBP-FlagIEP fusion protein (middle panel) or RmInt1 IEP mutant proteins (right panel) for 0, 5, 30, and 120 min. The arrows on the right indicate the products obtained: La, fully spliced lariat RNA; and, Pre, precursor RmInt1 ΔORF RNA. (B) Kinetic data, including additional time points, are plotted as the percentage lariat formation relative to the level of precursor RNA. Lines in black/squares represent the MBP-FlagIEP wild-type fusion protein; the RT-deficient mutant (MBP-FlagYAHH) is indicated by a broken black line/triangles; maturase domain mutants are indicated with a solid gray line/circles (MBP-FlagA354A355) or a broken line/diamonds (MBP-FlagA381). The experiment was repeated twice, with at least two independent purified fusion proteins for each mutant.

Mentions: As the fusion protein itself has RT activity, we investigated whether this protein could interact successfully with the intron ribozyme. Previous studies performed by our group revealed that RmInt1 efficiently self-spliced in vitro in the presence of high-magnesium (100 mM) buffer, but that these conditions gave rise to multiple products corresponding in size to the lariat intron-3′exon intermediate, and lariat or circle (double band) introns (Costa et al., 2006; Chillón et al., 2014). IEP-promoted splicing of a 906 nt ΔORF precursor RNA was monitored over time (Figure 3). Protein-assisted splicing of the ΔORF RNA precursor resulted in a single lariat RNA product (Figure 3A). A two-exponential model fitted the data well, with an initial fast reaction (>60% of lariat formation was completed in the first 15 min) followed by a phase of slow reactivity (Figure 3B). In the presence of MBP-FlagIEP wild-type, the percentage lariat formation reached 31%. IEP-promoted in vitro splicing was performed in different Mg2+ concentrations, but the results were not improved by increasing MgCl2 concentration beyond 5 mM (not shown). As previously reported, this low-magnesium buffer does not allow intron excision in the absence of the IEP (Figure 3, left panel). Hence, IEP assistance reduces magnesium requirements and accelerates lariat formation, preventing the production of secondary products. A two-phase reaction has also been reported for LtrA-assisted splicing, with a fast phase in which about 50–75% of the molecules react, followed by a slow reaction until the fraction spliced finally reaches 75–95%, depending on the conditions (Wank et al., 1999; Matsuura et al., 2001; Noah and Lambowitz, 2003; Cui et al., 2004).


Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles
RmInt1 splicing efficiency in vitro. (A) The left panel (lanes 1–2) shows the inability of RmInt1 to self-splice in vitro. Splicing reactions were carried out by incubating 32P-labeled, 905 nt RmInt1 ΔORF RNA with 5 mM MgCl2 for 0 and 300 min. The panels on the right (lanes 3–18) show RmInt1 IEP-assisted splicing, as evaluated by incubating a 32P-labeled ΔORF RNA precursor with different protein preparations in a buffer containing 5 mM MgCl2 for various times. The radioactively labeled precursor ΔORF RNA was incubated with a 50-fold excess of wild- type MBP-FlagIEP fusion protein (middle panel) or RmInt1 IEP mutant proteins (right panel) for 0, 5, 30, and 120 min. The arrows on the right indicate the products obtained: La, fully spliced lariat RNA; and, Pre, precursor RmInt1 ΔORF RNA. (B) Kinetic data, including additional time points, are plotted as the percentage lariat formation relative to the level of precursor RNA. Lines in black/squares represent the MBP-FlagIEP wild-type fusion protein; the RT-deficient mutant (MBP-FlagYAHH) is indicated by a broken black line/triangles; maturase domain mutants are indicated with a solid gray line/circles (MBP-FlagA354A355) or a broken line/diamonds (MBP-FlagA381). The experiment was repeated twice, with at least two independent purified fusion proteins for each mutant.
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Related In: Results  -  Collection

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Figure 3: RmInt1 splicing efficiency in vitro. (A) The left panel (lanes 1–2) shows the inability of RmInt1 to self-splice in vitro. Splicing reactions were carried out by incubating 32P-labeled, 905 nt RmInt1 ΔORF RNA with 5 mM MgCl2 for 0 and 300 min. The panels on the right (lanes 3–18) show RmInt1 IEP-assisted splicing, as evaluated by incubating a 32P-labeled ΔORF RNA precursor with different protein preparations in a buffer containing 5 mM MgCl2 for various times. The radioactively labeled precursor ΔORF RNA was incubated with a 50-fold excess of wild- type MBP-FlagIEP fusion protein (middle panel) or RmInt1 IEP mutant proteins (right panel) for 0, 5, 30, and 120 min. The arrows on the right indicate the products obtained: La, fully spliced lariat RNA; and, Pre, precursor RmInt1 ΔORF RNA. (B) Kinetic data, including additional time points, are plotted as the percentage lariat formation relative to the level of precursor RNA. Lines in black/squares represent the MBP-FlagIEP wild-type fusion protein; the RT-deficient mutant (MBP-FlagYAHH) is indicated by a broken black line/triangles; maturase domain mutants are indicated with a solid gray line/circles (MBP-FlagA354A355) or a broken line/diamonds (MBP-FlagA381). The experiment was repeated twice, with at least two independent purified fusion proteins for each mutant.
Mentions: As the fusion protein itself has RT activity, we investigated whether this protein could interact successfully with the intron ribozyme. Previous studies performed by our group revealed that RmInt1 efficiently self-spliced in vitro in the presence of high-magnesium (100 mM) buffer, but that these conditions gave rise to multiple products corresponding in size to the lariat intron-3′exon intermediate, and lariat or circle (double band) introns (Costa et al., 2006; Chillón et al., 2014). IEP-promoted splicing of a 906 nt ΔORF precursor RNA was monitored over time (Figure 3). Protein-assisted splicing of the ΔORF RNA precursor resulted in a single lariat RNA product (Figure 3A). A two-exponential model fitted the data well, with an initial fast reaction (>60% of lariat formation was completed in the first 15 min) followed by a phase of slow reactivity (Figure 3B). In the presence of MBP-FlagIEP wild-type, the percentage lariat formation reached 31%. IEP-promoted in vitro splicing was performed in different Mg2+ concentrations, but the results were not improved by increasing MgCl2 concentration beyond 5 mM (not shown). As previously reported, this low-magnesium buffer does not allow intron excision in the absence of the IEP (Figure 3, left panel). Hence, IEP assistance reduces magnesium requirements and accelerates lariat formation, preventing the production of secondary products. A two-phase reaction has also been reported for LtrA-assisted splicing, with a fast phase in which about 50–75% of the molecules react, followed by a slow reaction until the fraction spliced finally reaches 75–95%, depending on the conditions (Wank et al., 1999; Matsuura et al., 2001; Noah and Lambowitz, 2003; Cui et al., 2004).

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