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Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

Ponchon L, Catala M, Seijo B, El Khouri M, Dardel F, Nonin-Lecomte S, Tisné C - Nucleic Acids Res. (2013)

Bottom Line: RNA has emerged as a major player in many cellular processes.We show that this last application easily provides pure material for crystallographic studies.The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.

ABSTRACT
RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

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The use of His6-MS2 coat protein simplifies the RNA purification protocol. (A) Co-expression of AtRNA-mala/His6-MS2 coat protein in E. coli JM101 strain. Crude bacteria extracts before (lane 1) and after (lane 2) IPTG induction, and crude RNA minipreps (lane 3 and 4) were separated on a 16% SDS–PAGE gel and visualized by Coomassie Brilliant Blue staining and UV shadowing. Stroke indicates the control experiment: bacteria transformed by the vector with no insert. White boxes indicate the overexpressed AtRNA-mala and the MS2 coat protein. NiNTA elution (lane 5): the AtRNA-mala/His6-MS2 coat protein complex was eluted in the same fractions upon binding to NiNTA agarose column. The MS2 coat protein can then be digested using proteinase K (lane 6). The black triangle indicates the AtRNA-mala band and the grey triangle indicates the MS2 coat protein band. The molecular weight of protein standards is given in kilodalton on the left. (B) Comparison of benzonase resistance of the AtRNA-mala because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtRNA-mala. (C) Comparison of benzonase resistance of the AtmRNA because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtmRNA.
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gkt576-F5: The use of His6-MS2 coat protein simplifies the RNA purification protocol. (A) Co-expression of AtRNA-mala/His6-MS2 coat protein in E. coli JM101 strain. Crude bacteria extracts before (lane 1) and after (lane 2) IPTG induction, and crude RNA minipreps (lane 3 and 4) were separated on a 16% SDS–PAGE gel and visualized by Coomassie Brilliant Blue staining and UV shadowing. Stroke indicates the control experiment: bacteria transformed by the vector with no insert. White boxes indicate the overexpressed AtRNA-mala and the MS2 coat protein. NiNTA elution (lane 5): the AtRNA-mala/His6-MS2 coat protein complex was eluted in the same fractions upon binding to NiNTA agarose column. The MS2 coat protein can then be digested using proteinase K (lane 6). The black triangle indicates the AtRNA-mala band and the grey triangle indicates the MS2 coat protein band. The molecular weight of protein standards is given in kilodalton on the left. (B) Comparison of benzonase resistance of the AtRNA-mala because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtRNA-mala. (C) Comparison of benzonase resistance of the AtmRNA because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtmRNA.

Mentions: To facilitate the purification of the MS2 coat protein–tRNA-RNA complex, a polyhistidine tag was introduced into the loop region of the MS2 coat protein as previously reported (28). The resulting His6-MS2 construct was co-expressed with either AtRNA, AtRNA-mala or AtmRNA. In all cases, it was noticed that the MS2 coat protein only formed a dimer (Figure 3A, red chromatogram) and not pseudo-particles. Therefore, the polyhistidine tag prevents the MS2 coat protein from assembling into pseudo-particles. Nevertheless, this His6 tag offered a useful way to co-purify the RNA–protein complexes. Figure 5A shows that the AtRNA-His6-MS2 coat protein can be fixed to the Ni-NTA agarose resin and recovered by imidazole gradient elution. Remarkably, the overproduction of the His6-MS2 coat protein prevents the A. aeolicus AtmRNA from nucleolysis in E. coli despite its inability to form VLPs (Figure 5C).Figure 5.


Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

Ponchon L, Catala M, Seijo B, El Khouri M, Dardel F, Nonin-Lecomte S, Tisné C - Nucleic Acids Res. (2013)

The use of His6-MS2 coat protein simplifies the RNA purification protocol. (A) Co-expression of AtRNA-mala/His6-MS2 coat protein in E. coli JM101 strain. Crude bacteria extracts before (lane 1) and after (lane 2) IPTG induction, and crude RNA minipreps (lane 3 and 4) were separated on a 16% SDS–PAGE gel and visualized by Coomassie Brilliant Blue staining and UV shadowing. Stroke indicates the control experiment: bacteria transformed by the vector with no insert. White boxes indicate the overexpressed AtRNA-mala and the MS2 coat protein. NiNTA elution (lane 5): the AtRNA-mala/His6-MS2 coat protein complex was eluted in the same fractions upon binding to NiNTA agarose column. The MS2 coat protein can then be digested using proteinase K (lane 6). The black triangle indicates the AtRNA-mala band and the grey triangle indicates the MS2 coat protein band. The molecular weight of protein standards is given in kilodalton on the left. (B) Comparison of benzonase resistance of the AtRNA-mala because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtRNA-mala. (C) Comparison of benzonase resistance of the AtmRNA because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtmRNA.
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gkt576-F5: The use of His6-MS2 coat protein simplifies the RNA purification protocol. (A) Co-expression of AtRNA-mala/His6-MS2 coat protein in E. coli JM101 strain. Crude bacteria extracts before (lane 1) and after (lane 2) IPTG induction, and crude RNA minipreps (lane 3 and 4) were separated on a 16% SDS–PAGE gel and visualized by Coomassie Brilliant Blue staining and UV shadowing. Stroke indicates the control experiment: bacteria transformed by the vector with no insert. White boxes indicate the overexpressed AtRNA-mala and the MS2 coat protein. NiNTA elution (lane 5): the AtRNA-mala/His6-MS2 coat protein complex was eluted in the same fractions upon binding to NiNTA agarose column. The MS2 coat protein can then be digested using proteinase K (lane 6). The black triangle indicates the AtRNA-mala band and the grey triangle indicates the MS2 coat protein band. The molecular weight of protein standards is given in kilodalton on the left. (B) Comparison of benzonase resistance of the AtRNA-mala because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtRNA-mala. (C) Comparison of benzonase resistance of the AtmRNA because of co-expression with the MS2 protein (wild-type or His-tagged). RNA extracts in absence (−) or in presence (+) of benzonase in the lysis supernatant (sonication) were analysed by electrophoresis on a 16% SDS–PAGE gel and visualized by UV shadowing. The black triangle indicates the AtmRNA.
Mentions: To facilitate the purification of the MS2 coat protein–tRNA-RNA complex, a polyhistidine tag was introduced into the loop region of the MS2 coat protein as previously reported (28). The resulting His6-MS2 construct was co-expressed with either AtRNA, AtRNA-mala or AtmRNA. In all cases, it was noticed that the MS2 coat protein only formed a dimer (Figure 3A, red chromatogram) and not pseudo-particles. Therefore, the polyhistidine tag prevents the MS2 coat protein from assembling into pseudo-particles. Nevertheless, this His6 tag offered a useful way to co-purify the RNA–protein complexes. Figure 5A shows that the AtRNA-His6-MS2 coat protein can be fixed to the Ni-NTA agarose resin and recovered by imidazole gradient elution. Remarkably, the overproduction of the His6-MS2 coat protein prevents the A. aeolicus AtmRNA from nucleolysis in E. coli despite its inability to form VLPs (Figure 5C).Figure 5.

Bottom Line: RNA has emerged as a major player in many cellular processes.We show that this last application easily provides pure material for crystallographic studies.The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.

ABSTRACT
RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

Show MeSH
Related in: MedlinePlus