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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

Purified MBP-FlagIEP has reverse transcriptase activity in vitro. (A) The bar graphs show the reverse transcriptase activity of the MBP-FlagIEP fusion assayed with poly(rA)/oligo(dT)18 (black bar), or with poly(rA) [in the absence of oligo(dT)18, white bar]. The RT activity is expressed in counts per minute (cpm). The data shown are the means for at least three independent assays and three to seven independent MBP-FlagIEP preparations. Error bars represent the standard errors. (B) The RT activity of several mutant proteins was evaluated relative to the wild-type fusion protein. Note that the Y axis is represented in logarithmic scale. RT values were derived from at least three experimental replicates, with three different protein preparations used for each mutant. Error bars correspond to the standard errors.
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Figure 2: Purified MBP-FlagIEP has reverse transcriptase activity in vitro. (A) The bar graphs show the reverse transcriptase activity of the MBP-FlagIEP fusion assayed with poly(rA)/oligo(dT)18 (black bar), or with poly(rA) [in the absence of oligo(dT)18, white bar]. The RT activity is expressed in counts per minute (cpm). The data shown are the means for at least three independent assays and three to seven independent MBP-FlagIEP preparations. Error bars represent the standard errors. (B) The RT activity of several mutant proteins was evaluated relative to the wild-type fusion protein. Note that the Y axis is represented in logarithmic scale. RT values were derived from at least three experimental replicates, with three different protein preparations used for each mutant. Error bars correspond to the standard errors.

Mentions: We assessed the functionality of the protein, by carrying out reverse transcriptase assays with the exogenous substrate poly(rA)/oligo(dT)18 (Figure 2A). The MBP-FlagIEP fusion protein displayed significant RT activity in the presence of the RNA substrate and the DNA primer (8.6 × 105 cpm), whereas the absence of oligo(dT)18 resulted in much lower levels of cDNA synthesis (2 × 103 cpm). As a control, we also investigated three other fusion proteins in which conserved residues of the RT and maturase domains had been mutated (Figure 2B). As expected, cDNA synthesis was abolished in the RT-deficient YAHH mutant (0.3% wild-type), in which two essential aspartate residues in RT domain 5 were replaced with histidines (Muñoz-Adelantado et al., 2003). The YYAA maturase- mutant, in which two conserved tyrosine residues at positions 354–355 in the RGWXNYY maturase motif were replaced with two alanine residues, had very low levels of RT activity (1.7% wild-type). In addition, the K381A mutant, with a substitution of the lysine 381 residue in the conserved maturase R(K/R)XK motif decreasing intron excision rates to 30% those for the wild type (Molina-Sánchez et al., 2006, 2010), had low levels of RT activity (22% wild-type). These results suggest that these residues might be relevant for the substrate docking required for cDNA synthesis, to somewhat different degrees. Thus, MBP-FlagIEP was stable and active when assayed alone in the absence of the intron RNA.


Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles
Purified MBP-FlagIEP has reverse transcriptase activity in vitro. (A) The bar graphs show the reverse transcriptase activity of the MBP-FlagIEP fusion assayed with poly(rA)/oligo(dT)18 (black bar), or with poly(rA) [in the absence of oligo(dT)18, white bar]. The RT activity is expressed in counts per minute (cpm). The data shown are the means for at least three independent assays and three to seven independent MBP-FlagIEP preparations. Error bars represent the standard errors. (B) The RT activity of several mutant proteins was evaluated relative to the wild-type fusion protein. Note that the Y axis is represented in logarithmic scale. RT values were derived from at least three experimental replicates, with three different protein preparations used for each mutant. Error bars correspond to the standard errors.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037169&req=5

Figure 2: Purified MBP-FlagIEP has reverse transcriptase activity in vitro. (A) The bar graphs show the reverse transcriptase activity of the MBP-FlagIEP fusion assayed with poly(rA)/oligo(dT)18 (black bar), or with poly(rA) [in the absence of oligo(dT)18, white bar]. The RT activity is expressed in counts per minute (cpm). The data shown are the means for at least three independent assays and three to seven independent MBP-FlagIEP preparations. Error bars represent the standard errors. (B) The RT activity of several mutant proteins was evaluated relative to the wild-type fusion protein. Note that the Y axis is represented in logarithmic scale. RT values were derived from at least three experimental replicates, with three different protein preparations used for each mutant. Error bars correspond to the standard errors.
Mentions: We assessed the functionality of the protein, by carrying out reverse transcriptase assays with the exogenous substrate poly(rA)/oligo(dT)18 (Figure 2A). The MBP-FlagIEP fusion protein displayed significant RT activity in the presence of the RNA substrate and the DNA primer (8.6 × 105 cpm), whereas the absence of oligo(dT)18 resulted in much lower levels of cDNA synthesis (2 × 103 cpm). As a control, we also investigated three other fusion proteins in which conserved residues of the RT and maturase domains had been mutated (Figure 2B). As expected, cDNA synthesis was abolished in the RT-deficient YAHH mutant (0.3% wild-type), in which two essential aspartate residues in RT domain 5 were replaced with histidines (Muñoz-Adelantado et al., 2003). The YYAA maturase- mutant, in which two conserved tyrosine residues at positions 354–355 in the RGWXNYY maturase motif were replaced with two alanine residues, had very low levels of RT activity (1.7% wild-type). In addition, the K381A mutant, with a substitution of the lysine 381 residue in the conserved maturase R(K/R)XK motif decreasing intron excision rates to 30% those for the wild type (Molina-Sánchez et al., 2006, 2010), had low levels of RT activity (22% wild-type). These results suggest that these residues might be relevant for the substrate docking required for cDNA synthesis, to somewhat different degrees. Thus, MBP-FlagIEP was stable and active when assayed alone in the absence of the intron RNA.

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