Limits...
Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay.

Kolesnikova O, Back R, Graille M, Séraphin B - Nucleic Acids Res. (2013)

Bottom Line: In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA).We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein.This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum.

View Article: PubMed Central - PubMed

Affiliation: Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 67404 Illkirch, France, Ecole Polytechnique, Laboratoire de Biochimie, CNRS UMR7654, 91128 Palaiseau Cedex, France and Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC), CNRS, UMR8619, Bat 430, Université Paris Sud, 91405 Orsay Cedex, France.

ABSTRACT
In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the auto-regulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3.

Show MeSH

Related in: MedlinePlus

The RB motif of Edc3 is needed for interaction with Rps28a protein. (A) Schematic representation of the operon constructs used for expressing variants of S. cerevisiae Edc3 and Rps28a in E. coli. A 6His tag was fused to the N-terminus of Rps28a. Numbers above the schematic representation of the proteins correspond to the amino acid boundaries. Pattern codes for Edc3 domains are as in Figure 1. (B) Coomassie blue-stained Tris–Tricine–SDS–PAGE showing co-purification of recombinant Edc3 and Rps28a proteins. Supernatants of lysed E. coli cells expressing recombinant yeast 6His-Rps28a and Edc3 protein fragments were incubated with Ni-NTA beads and subsequently washed before elution of bound proteins with imidazole. S, supernatant of lysed cells; F, flow through; E, elution. (C) Beta-galactosidase activity measurements to monitor interactions in the two-hybrid assay. Full-length wild-type Edc3 and derivative Edc3ΔRB were fused to the GAL4 activation domain, whereas Dcp2, Dhh1 and Rps28 proteins were fused to the GAL4 DNA-binding domain. In each case, the matching vector was used as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814365&req=5

gkt607-F2: The RB motif of Edc3 is needed for interaction with Rps28a protein. (A) Schematic representation of the operon constructs used for expressing variants of S. cerevisiae Edc3 and Rps28a in E. coli. A 6His tag was fused to the N-terminus of Rps28a. Numbers above the schematic representation of the proteins correspond to the amino acid boundaries. Pattern codes for Edc3 domains are as in Figure 1. (B) Coomassie blue-stained Tris–Tricine–SDS–PAGE showing co-purification of recombinant Edc3 and Rps28a proteins. Supernatants of lysed E. coli cells expressing recombinant yeast 6His-Rps28a and Edc3 protein fragments were incubated with Ni-NTA beads and subsequently washed before elution of bound proteins with imidazole. S, supernatant of lysed cells; F, flow through; E, elution. (C) Beta-galactosidase activity measurements to monitor interactions in the two-hybrid assay. Full-length wild-type Edc3 and derivative Edc3ΔRB were fused to the GAL4 activation domain, whereas Dcp2, Dhh1 and Rps28 proteins were fused to the GAL4 DNA-binding domain. In each case, the matching vector was used as negative control.

Mentions: Experimental evidences supporting an interaction between Edc3 and Rps28 were derived from two-hydrid and co-immunoprecipitation analyses (19). These data did not indicate whether Rps28 and Edc3 interact directly, possibly in a manner stabilized by protein and/or RNA partners, or whether their interaction was exclusively bridged by other factor(s). To study the role of the newly defined Edc3 motif, we, thus, first sought to develop a more direct protein interaction assays. For this purpose, we constructed plasmids to overexpress Edc3, either alone or together with Rps28a in Escherichia coli (Figure 2A). Although plasmids encoding full-length Edc3 produced insoluble protein (data not shown), a construct carrying an operon encoding a 6His-tagged Rps28 protein and Edc3 lacking its C-terminal YjeF-N domain [Edc3(1–277)] expressed both proteins in a soluble form (Figure 2B). Moreover, affinity chromatography on Ni-NTA beads demonstrated a co-purification of recombinant Rps28 and Edc3 (Figure 2B). A control construct lacking the 6His-Rps28 demonstrated that Edc3(1–277) did not bind non-specifically to Ni-NTA beads. These results indicate that Rps28 and Edc3 interact directly forming a stable complex without requirement for additional RNA or protein partners, and further that the YjeF-N domain of Edc3 is not required for this interaction.Figure 2.


Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay.

Kolesnikova O, Back R, Graille M, Séraphin B - Nucleic Acids Res. (2013)

The RB motif of Edc3 is needed for interaction with Rps28a protein. (A) Schematic representation of the operon constructs used for expressing variants of S. cerevisiae Edc3 and Rps28a in E. coli. A 6His tag was fused to the N-terminus of Rps28a. Numbers above the schematic representation of the proteins correspond to the amino acid boundaries. Pattern codes for Edc3 domains are as in Figure 1. (B) Coomassie blue-stained Tris–Tricine–SDS–PAGE showing co-purification of recombinant Edc3 and Rps28a proteins. Supernatants of lysed E. coli cells expressing recombinant yeast 6His-Rps28a and Edc3 protein fragments were incubated with Ni-NTA beads and subsequently washed before elution of bound proteins with imidazole. S, supernatant of lysed cells; F, flow through; E, elution. (C) Beta-galactosidase activity measurements to monitor interactions in the two-hybrid assay. Full-length wild-type Edc3 and derivative Edc3ΔRB were fused to the GAL4 activation domain, whereas Dcp2, Dhh1 and Rps28 proteins were fused to the GAL4 DNA-binding domain. In each case, the matching vector was used as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814365&req=5

gkt607-F2: The RB motif of Edc3 is needed for interaction with Rps28a protein. (A) Schematic representation of the operon constructs used for expressing variants of S. cerevisiae Edc3 and Rps28a in E. coli. A 6His tag was fused to the N-terminus of Rps28a. Numbers above the schematic representation of the proteins correspond to the amino acid boundaries. Pattern codes for Edc3 domains are as in Figure 1. (B) Coomassie blue-stained Tris–Tricine–SDS–PAGE showing co-purification of recombinant Edc3 and Rps28a proteins. Supernatants of lysed E. coli cells expressing recombinant yeast 6His-Rps28a and Edc3 protein fragments were incubated with Ni-NTA beads and subsequently washed before elution of bound proteins with imidazole. S, supernatant of lysed cells; F, flow through; E, elution. (C) Beta-galactosidase activity measurements to monitor interactions in the two-hybrid assay. Full-length wild-type Edc3 and derivative Edc3ΔRB were fused to the GAL4 activation domain, whereas Dcp2, Dhh1 and Rps28 proteins were fused to the GAL4 DNA-binding domain. In each case, the matching vector was used as negative control.
Mentions: Experimental evidences supporting an interaction between Edc3 and Rps28 were derived from two-hydrid and co-immunoprecipitation analyses (19). These data did not indicate whether Rps28 and Edc3 interact directly, possibly in a manner stabilized by protein and/or RNA partners, or whether their interaction was exclusively bridged by other factor(s). To study the role of the newly defined Edc3 motif, we, thus, first sought to develop a more direct protein interaction assays. For this purpose, we constructed plasmids to overexpress Edc3, either alone or together with Rps28a in Escherichia coli (Figure 2A). Although plasmids encoding full-length Edc3 produced insoluble protein (data not shown), a construct carrying an operon encoding a 6His-tagged Rps28 protein and Edc3 lacking its C-terminal YjeF-N domain [Edc3(1–277)] expressed both proteins in a soluble form (Figure 2B). Moreover, affinity chromatography on Ni-NTA beads demonstrated a co-purification of recombinant Rps28 and Edc3 (Figure 2B). A control construct lacking the 6His-Rps28 demonstrated that Edc3(1–277) did not bind non-specifically to Ni-NTA beads. These results indicate that Rps28 and Edc3 interact directly forming a stable complex without requirement for additional RNA or protein partners, and further that the YjeF-N domain of Edc3 is not required for this interaction.Figure 2.

Bottom Line: In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA).We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein.This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum.

View Article: PubMed Central - PubMed

Affiliation: Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 67404 Illkirch, France, Ecole Polytechnique, Laboratoire de Biochimie, CNRS UMR7654, 91128 Palaiseau Cedex, France and Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC), CNRS, UMR8619, Bat 430, Université Paris Sud, 91405 Orsay Cedex, France.

ABSTRACT
In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the auto-regulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3.

Show MeSH
Related in: MedlinePlus