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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

The MBP-FlagIEP fusion protein is functional in vivo. (A) The scaled scheme shows the constructs used as intron donors. pKG4_MAL is a derivative of pKGEMA4 in which the IEP has been replaced by the fusion protein MBP-FlagIEP. PKm, kanamycin promoter; black arrows identify the IEP; an open box flanked by gray lines corresponds to the intron ΔORF and short −20/+5 exons, respectively. (B) A homing assay is shown in the upper panel. The donor constructs indicated above were used to transform S. meliloti RMO17, an intron-less strain, containing the recipient plasmid pJB0.6LAG. The plasmid pools were analyzed by Southern hybridization with a DNA intron probe. An intron donor plasmid harboring a mutation in the catalytic domain of the ribozyme (pKG4dV) or a recipient plasmid lacking the recognition DNA target region (pJBΔ129) was used as a control. The lower panel corresponds to quantification of the retrohoming efficiency expressed as a percentage relative to that for the pKGEMA4 construct. The data shown are the means for at least three independent colonies ± standard errors.
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Figure 1: The MBP-FlagIEP fusion protein is functional in vivo. (A) The scaled scheme shows the constructs used as intron donors. pKG4_MAL is a derivative of pKGEMA4 in which the IEP has been replaced by the fusion protein MBP-FlagIEP. PKm, kanamycin promoter; black arrows identify the IEP; an open box flanked by gray lines corresponds to the intron ΔORF and short −20/+5 exons, respectively. (B) A homing assay is shown in the upper panel. The donor constructs indicated above were used to transform S. meliloti RMO17, an intron-less strain, containing the recipient plasmid pJB0.6LAG. The plasmid pools were analyzed by Southern hybridization with a DNA intron probe. An intron donor plasmid harboring a mutation in the catalytic domain of the ribozyme (pKG4dV) or a recipient plasmid lacking the recognition DNA target region (pJBΔ129) was used as a control. The lower panel corresponds to quantification of the retrohoming efficiency expressed as a percentage relative to that for the pKGEMA4 construct. The data shown are the means for at least three independent colonies ± standard errors.

Mentions: The IEPs of group II introns are proteins with a high proportion of positively charged amino acids and an alkaline isoelectric point, and their stability and solubility are, therefore, usually low (Mohr et al., 2013; Zhao and Pyle, 2016). We overcame these problems by producing the RmInt1 IEP as a fusion with the maltose binding protein (pMAL, New England Biolabs). The construct encoded the maltose binding protein (malE, MBP) followed by the Flag epitope, all in-frame with the N-terminal region of the RmInt1 intron ORF. We assessed the competence of the MBP-FlagIEP fusion protein for assisting the in vivo activities of RmInt1 intron RNA, by carrying out retrohoming assays with a two-plasmid (donor/recipient) system (Martínez-Abarca et al., 2004). We used an intron donor plasmid derived from pKGEMA4 (Nisa-Martínez et al., 2007) and encoding the fusion protein under the control of the kanamycin promoter (PKm) and upstream from the RmInt1 ΔORF ribozyme flanked by exon sequences −20/+5 (Figure 1A). S. meliloti RMO17, an intronless strain, harboring the recipient plasmid pJB0.6LAG was transformed with the pKG4_MALFlagIEP intron donor plasmid (Figure 1B, lane 1). Homing efficiency was slightly lower (21%) than for the wild-type protein construct, pKGEMA4 (lane 2). The MBP-IEP fusion protein therefore seemed to be fully functional in vivo.


Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles
The MBP-FlagIEP fusion protein is functional in vivo. (A) The scaled scheme shows the constructs used as intron donors. pKG4_MAL is a derivative of pKGEMA4 in which the IEP has been replaced by the fusion protein MBP-FlagIEP. PKm, kanamycin promoter; black arrows identify the IEP; an open box flanked by gray lines corresponds to the intron ΔORF and short −20/+5 exons, respectively. (B) A homing assay is shown in the upper panel. The donor constructs indicated above were used to transform S. meliloti RMO17, an intron-less strain, containing the recipient plasmid pJB0.6LAG. The plasmid pools were analyzed by Southern hybridization with a DNA intron probe. An intron donor plasmid harboring a mutation in the catalytic domain of the ribozyme (pKG4dV) or a recipient plasmid lacking the recognition DNA target region (pJBΔ129) was used as a control. The lower panel corresponds to quantification of the retrohoming efficiency expressed as a percentage relative to that for the pKGEMA4 construct. The data shown are the means for at least three independent colonies ± standard errors.
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Figure 1: The MBP-FlagIEP fusion protein is functional in vivo. (A) The scaled scheme shows the constructs used as intron donors. pKG4_MAL is a derivative of pKGEMA4 in which the IEP has been replaced by the fusion protein MBP-FlagIEP. PKm, kanamycin promoter; black arrows identify the IEP; an open box flanked by gray lines corresponds to the intron ΔORF and short −20/+5 exons, respectively. (B) A homing assay is shown in the upper panel. The donor constructs indicated above were used to transform S. meliloti RMO17, an intron-less strain, containing the recipient plasmid pJB0.6LAG. The plasmid pools were analyzed by Southern hybridization with a DNA intron probe. An intron donor plasmid harboring a mutation in the catalytic domain of the ribozyme (pKG4dV) or a recipient plasmid lacking the recognition DNA target region (pJBΔ129) was used as a control. The lower panel corresponds to quantification of the retrohoming efficiency expressed as a percentage relative to that for the pKGEMA4 construct. The data shown are the means for at least three independent colonies ± standard errors.
Mentions: The IEPs of group II introns are proteins with a high proportion of positively charged amino acids and an alkaline isoelectric point, and their stability and solubility are, therefore, usually low (Mohr et al., 2013; Zhao and Pyle, 2016). We overcame these problems by producing the RmInt1 IEP as a fusion with the maltose binding protein (pMAL, New England Biolabs). The construct encoded the maltose binding protein (malE, MBP) followed by the Flag epitope, all in-frame with the N-terminal region of the RmInt1 intron ORF. We assessed the competence of the MBP-FlagIEP fusion protein for assisting the in vivo activities of RmInt1 intron RNA, by carrying out retrohoming assays with a two-plasmid (donor/recipient) system (Martínez-Abarca et al., 2004). We used an intron donor plasmid derived from pKGEMA4 (Nisa-Martínez et al., 2007) and encoding the fusion protein under the control of the kanamycin promoter (PKm) and upstream from the RmInt1 ΔORF ribozyme flanked by exon sequences −20/+5 (Figure 1A). S. meliloti RMO17, an intronless strain, harboring the recipient plasmid pJB0.6LAG was transformed with the pKG4_MALFlagIEP intron donor plasmid (Figure 1B, lane 1). Homing efficiency was slightly lower (21%) than for the wild-type protein construct, pKGEMA4 (lane 2). The MBP-IEP fusion protein therefore seemed to be fully functional in vivo.

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