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The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

Wegrecki M, Marcin W, Neira JL, Bravo J - PLoS ONE (2015)

Bottom Line: This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat.The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding.Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, c/ Jaime Roig 11, 46010 Valencia, Spain.

ABSTRACT
Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

No MeSH data available.


Related in: MedlinePlus

ChErb1Ct (residues 432–801) binds RNA in vitro.(a) Coomassie-stained SDS-PAGE showing the binding of Erb1 β-propeller from Ch. thermophilum to polyU agarose beads. (b) The saturation of the binding is visible upon addition of 0.1 mg/ml or 1 mg/ml of free polyuridilic acid. (c) The binding is still detectable upon addition of 1 mg/ml of heparin to the binding buffer. (a) (b) and (c) 1: Input, 2: Wash, 3: Elution; (d) Fluorescence spectra of ChErb1432-801 alone (in black) and with 5μM poly(U) (blue) obtained by excitation at 280 nm. The spectra were acquired at 25°C with 1.5 μM of ChErb1432-801. The green line at the bottom of the spectra is the emission spectra of polyU at a concentration of 5 μM. (e) Titration curve obtained from the emission fluorescence intensity at 315 nm. The line is the fitting to Eq (1). (f) Association and dissociation curves of 15nt-poly(U) with different concentrations of ChErb1432-801 measured by biolayer interferometry.
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pone.0123463.g008: ChErb1Ct (residues 432–801) binds RNA in vitro.(a) Coomassie-stained SDS-PAGE showing the binding of Erb1 β-propeller from Ch. thermophilum to polyU agarose beads. (b) The saturation of the binding is visible upon addition of 0.1 mg/ml or 1 mg/ml of free polyuridilic acid. (c) The binding is still detectable upon addition of 1 mg/ml of heparin to the binding buffer. (a) (b) and (c) 1: Input, 2: Wash, 3: Elution; (d) Fluorescence spectra of ChErb1432-801 alone (in black) and with 5μM poly(U) (blue) obtained by excitation at 280 nm. The spectra were acquired at 25°C with 1.5 μM of ChErb1432-801. The green line at the bottom of the spectra is the emission spectra of polyU at a concentration of 5 μM. (e) Titration curve obtained from the emission fluorescence intensity at 315 nm. The line is the fitting to Eq (1). (f) Association and dissociation curves of 15nt-poly(U) with different concentrations of ChErb1432-801 measured by biolayer interferometry.

Mentions: Initially, we checked the affinity of the β-propeller of Erb1 for RNA in vitro using poly(U) agarose beads. Our first attempts to perform in vitro binding assays failed because the C-terminal domain of Erb1 (residues 416–807) from yeast expressed poorly in E. coli and rapidly degraded during purification. We decided to try whether the same domain from a thermophile would be stable enough to carry out the experiments. Finally, a truncated Erb1 from Chaetomium thermophilum, comprising residues 432–801 (ChErb1432-801), was used for the assays due to its enhanced stability and higher expression levels in E. coli.Since the sequence of the domain is well conserved between S. cerevisiae and C. thermophilum, including the basic residues from the putative RNA-binding area (shown with green boxes in Fig 1c), we consider ChErb1432-801 to be suitable for validation of our findings based on Erb1416-807 structure from yeast. As shown in the Fig 8a the propeller appeared in the eluate from poly(U) beads. To investigate whether the interaction occurred through a well-defined surface that could be saturated, we incubated the protein with free poly(U) before it was loaded on the beads. The amount of the protein that could stably bind the poly(U) beads decreased in presence of 0.1mg/ml and 1mg/ml free poly(U), indicating saturation of the binding site (Fig 8b). The binding affinity was good because it was still detectable even when heparin was added to the reaction mixture (Fig 8c). Fluorescence experiments were carried out in order to estimate the binding affinity. Emission spectrum of intact ChErb1432-801 shows a maximum wavelength at 340 nm (Fig 8d), indicating that the tryptophans in the structure are partially buried, as shown in the X-ray structure. Therefore, if the RNA binding occurred in the proximity of the indole moiety, we should expect changes in the fluorescence spectra upon addition of polyU, and therefore we could use these changes to measure the affinity constants by using Eq (1). We acquired spectra at growing concentrations of polyU (Fig 8e), and the changes in the fluorescence emission spectra at 315 nm allowed us to determine the constant (similar changes were observed at other wavelengths either by excitation at 280 or 295 nm). The apparent KD of the complex was 3 ± 2 μM. The fact that an exposed tryptophan (Trp682, red oval in the Fig 7a) is at the vicinity of the positively charged stretch (likely to participate in RNA binding), provides a good indication that the interaction takes place through the proposed area and explains the change in fluorescence upon binding of the nucleic acid. We also calculated binding affinity between ChErb1432-801 and a 15 nucleotide-long biotinylated poly(U) using biolayer interferometry. The dissociation constant (KD) was 0.17 μM (Fig 8f).


The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

Wegrecki M, Marcin W, Neira JL, Bravo J - PLoS ONE (2015)

ChErb1Ct (residues 432–801) binds RNA in vitro.(a) Coomassie-stained SDS-PAGE showing the binding of Erb1 β-propeller from Ch. thermophilum to polyU agarose beads. (b) The saturation of the binding is visible upon addition of 0.1 mg/ml or 1 mg/ml of free polyuridilic acid. (c) The binding is still detectable upon addition of 1 mg/ml of heparin to the binding buffer. (a) (b) and (c) 1: Input, 2: Wash, 3: Elution; (d) Fluorescence spectra of ChErb1432-801 alone (in black) and with 5μM poly(U) (blue) obtained by excitation at 280 nm. The spectra were acquired at 25°C with 1.5 μM of ChErb1432-801. The green line at the bottom of the spectra is the emission spectra of polyU at a concentration of 5 μM. (e) Titration curve obtained from the emission fluorescence intensity at 315 nm. The line is the fitting to Eq (1). (f) Association and dissociation curves of 15nt-poly(U) with different concentrations of ChErb1432-801 measured by biolayer interferometry.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400149&req=5

pone.0123463.g008: ChErb1Ct (residues 432–801) binds RNA in vitro.(a) Coomassie-stained SDS-PAGE showing the binding of Erb1 β-propeller from Ch. thermophilum to polyU agarose beads. (b) The saturation of the binding is visible upon addition of 0.1 mg/ml or 1 mg/ml of free polyuridilic acid. (c) The binding is still detectable upon addition of 1 mg/ml of heparin to the binding buffer. (a) (b) and (c) 1: Input, 2: Wash, 3: Elution; (d) Fluorescence spectra of ChErb1432-801 alone (in black) and with 5μM poly(U) (blue) obtained by excitation at 280 nm. The spectra were acquired at 25°C with 1.5 μM of ChErb1432-801. The green line at the bottom of the spectra is the emission spectra of polyU at a concentration of 5 μM. (e) Titration curve obtained from the emission fluorescence intensity at 315 nm. The line is the fitting to Eq (1). (f) Association and dissociation curves of 15nt-poly(U) with different concentrations of ChErb1432-801 measured by biolayer interferometry.
Mentions: Initially, we checked the affinity of the β-propeller of Erb1 for RNA in vitro using poly(U) agarose beads. Our first attempts to perform in vitro binding assays failed because the C-terminal domain of Erb1 (residues 416–807) from yeast expressed poorly in E. coli and rapidly degraded during purification. We decided to try whether the same domain from a thermophile would be stable enough to carry out the experiments. Finally, a truncated Erb1 from Chaetomium thermophilum, comprising residues 432–801 (ChErb1432-801), was used for the assays due to its enhanced stability and higher expression levels in E. coli.Since the sequence of the domain is well conserved between S. cerevisiae and C. thermophilum, including the basic residues from the putative RNA-binding area (shown with green boxes in Fig 1c), we consider ChErb1432-801 to be suitable for validation of our findings based on Erb1416-807 structure from yeast. As shown in the Fig 8a the propeller appeared in the eluate from poly(U) beads. To investigate whether the interaction occurred through a well-defined surface that could be saturated, we incubated the protein with free poly(U) before it was loaded on the beads. The amount of the protein that could stably bind the poly(U) beads decreased in presence of 0.1mg/ml and 1mg/ml free poly(U), indicating saturation of the binding site (Fig 8b). The binding affinity was good because it was still detectable even when heparin was added to the reaction mixture (Fig 8c). Fluorescence experiments were carried out in order to estimate the binding affinity. Emission spectrum of intact ChErb1432-801 shows a maximum wavelength at 340 nm (Fig 8d), indicating that the tryptophans in the structure are partially buried, as shown in the X-ray structure. Therefore, if the RNA binding occurred in the proximity of the indole moiety, we should expect changes in the fluorescence spectra upon addition of polyU, and therefore we could use these changes to measure the affinity constants by using Eq (1). We acquired spectra at growing concentrations of polyU (Fig 8e), and the changes in the fluorescence emission spectra at 315 nm allowed us to determine the constant (similar changes were observed at other wavelengths either by excitation at 280 or 295 nm). The apparent KD of the complex was 3 ± 2 μM. The fact that an exposed tryptophan (Trp682, red oval in the Fig 7a) is at the vicinity of the positively charged stretch (likely to participate in RNA binding), provides a good indication that the interaction takes place through the proposed area and explains the change in fluorescence upon binding of the nucleic acid. We also calculated binding affinity between ChErb1432-801 and a 15 nucleotide-long biotinylated poly(U) using biolayer interferometry. The dissociation constant (KD) was 0.17 μM (Fig 8f).

Bottom Line: This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat.The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding.Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, c/ Jaime Roig 11, 46010 Valencia, Spain.

ABSTRACT
Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

No MeSH data available.


Related in: MedlinePlus