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The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus.

Senissar M, Le Saux A, Belgareh-Touzé N, Adam C, Banroques J, Tanner NK - Nucleic Acids Res. (2014)

Bottom Line: In addition, we show that Ded1 is genetically linked to these factors.Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes.We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.

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Ded1 physically interacted with the cap-associated factors. (A) Ded1 was bound to protein A Sepharose beads crosslinked to IgG against Ded1, incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was subsequently stained with Coomassie blue. Note that Cbp80 was weakly stained, but it was clearly visible on the original gel. The ‘nonspec’ lane was a mix of all the purified proteins incubated with the resin in the absence of Ded1. (B) MalE-Cbp80 was bound to amylose resin beads (Biolabs), incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was analyzed by western blot with IgG against Ded1, Cbp80 and the His6 tag of Cbp20 (Roche). Two identical gels were used to probe for Ded1 and Cbp20 because different secondary antibodies were used. Aliquots of the purified proteins (0.1 μg) are shown as markers. Note the Cbp20 protein migrated slower in the binding assay lanes due to the higher salt concentrations. (C) Purified GST-eIF4G1 fusion fragments bound to glutathione agarose beads, were incubated with Ded1, washed, eluted with reduced glutathione and separated by 10% SDS PAGE, which was subsequently stained with Coomassie blue. *The exception was the eIF4G-His6 fragment 387–952 that was retained by Ded1 bound to IgG-protein A Sepharose beads as in panel (A). Note that the 882–952 fragment gave a weak signal that was clearly visible on the original gel. For unknown reasons, the 1–387-GST fragment migrated as an aberrantly large protein. (D) The different fragments of eIF4G1 that were used in these experiments that show the locations of the previously identified binding motifs. RNA1–3 motifs bind the mRNA; motifs Pab1, eIF4E and eIF4A form direct protein–protein interactions with the corresponding proteins; and the solid line in the eIF4A-binding motif is the MIF4G/HEAT repeat domain that forms a protein–protein interaction surface. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
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Figure 5: Ded1 physically interacted with the cap-associated factors. (A) Ded1 was bound to protein A Sepharose beads crosslinked to IgG against Ded1, incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was subsequently stained with Coomassie blue. Note that Cbp80 was weakly stained, but it was clearly visible on the original gel. The ‘nonspec’ lane was a mix of all the purified proteins incubated with the resin in the absence of Ded1. (B) MalE-Cbp80 was bound to amylose resin beads (Biolabs), incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was analyzed by western blot with IgG against Ded1, Cbp80 and the His6 tag of Cbp20 (Roche). Two identical gels were used to probe for Ded1 and Cbp20 because different secondary antibodies were used. Aliquots of the purified proteins (0.1 μg) are shown as markers. Note the Cbp20 protein migrated slower in the binding assay lanes due to the higher salt concentrations. (C) Purified GST-eIF4G1 fusion fragments bound to glutathione agarose beads, were incubated with Ded1, washed, eluted with reduced glutathione and separated by 10% SDS PAGE, which was subsequently stained with Coomassie blue. *The exception was the eIF4G-His6 fragment 387–952 that was retained by Ded1 bound to IgG-protein A Sepharose beads as in panel (A). Note that the 882–952 fragment gave a weak signal that was clearly visible on the original gel. For unknown reasons, the 1–387-GST fragment migrated as an aberrantly large protein. (D) The different fragments of eIF4G1 that were used in these experiments that show the locations of the previously identified binding motifs. RNA1–3 motifs bind the mRNA; motifs Pab1, eIF4E and eIF4A form direct protein–protein interactions with the corresponding proteins; and the solid line in the eIF4A-binding motif is the MIF4G/HEAT repeat domain that forms a protein–protein interaction surface. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).

Mentions: The preceding experiments demonstrated direct physical interactions between Ded1 and cap-binding proteins. However, it was possible that some factors were indirectly associated with Ded1 through their interactions with the other proteins. Therefore, we individually tested various purified proteins for their ability to be retained by Ded1 fixed to Ded1-IgG Sepharose beads (Figure 5A). As a control, we incubated a mixture of the proteins with the beads in the absence of Ded1. Only Gle1 and Nab2 showed a weak nonspecific binding. The beads retained all of the indicated cap-associated factors in the presence of Ded1 except eIF4A. Both Pab1 and Cbp80 showed faint signals, but their presence was verified by western blot analysis. Ded1 bound to the beads also retained Gle1. This was consistent with previous observations that Gle1 physically and genetically interacts with Ded1 (75). As a control, we used the sec63 domain of Brr2 that is associated with protein–protein interactions (80); no interactions were detected, which indicated that only specific proteins were retained by Ded1. To verify the interactions between Ded1 and Cbp80, we bound a fusion construct between Mal-E and Cbp80 on amylose beads and tested for the ability to retain Ded1. Both Ded1 and Cbp20 were detected by western blot analysis in fractions containing MalE-Cbp80, but not in those lacking the fusion protein (Figure 5B). The Mal-E protein bound alone to the beads was unable to retain the proteins, which demonstrated that the interactions were mediated through Cbp80 (data not shown).


The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus.

Senissar M, Le Saux A, Belgareh-Touzé N, Adam C, Banroques J, Tanner NK - Nucleic Acids Res. (2014)

Ded1 physically interacted with the cap-associated factors. (A) Ded1 was bound to protein A Sepharose beads crosslinked to IgG against Ded1, incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was subsequently stained with Coomassie blue. Note that Cbp80 was weakly stained, but it was clearly visible on the original gel. The ‘nonspec’ lane was a mix of all the purified proteins incubated with the resin in the absence of Ded1. (B) MalE-Cbp80 was bound to amylose resin beads (Biolabs), incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was analyzed by western blot with IgG against Ded1, Cbp80 and the His6 tag of Cbp20 (Roche). Two identical gels were used to probe for Ded1 and Cbp20 because different secondary antibodies were used. Aliquots of the purified proteins (0.1 μg) are shown as markers. Note the Cbp20 protein migrated slower in the binding assay lanes due to the higher salt concentrations. (C) Purified GST-eIF4G1 fusion fragments bound to glutathione agarose beads, were incubated with Ded1, washed, eluted with reduced glutathione and separated by 10% SDS PAGE, which was subsequently stained with Coomassie blue. *The exception was the eIF4G-His6 fragment 387–952 that was retained by Ded1 bound to IgG-protein A Sepharose beads as in panel (A). Note that the 882–952 fragment gave a weak signal that was clearly visible on the original gel. For unknown reasons, the 1–387-GST fragment migrated as an aberrantly large protein. (D) The different fragments of eIF4G1 that were used in these experiments that show the locations of the previously identified binding motifs. RNA1–3 motifs bind the mRNA; motifs Pab1, eIF4E and eIF4A form direct protein–protein interactions with the corresponding proteins; and the solid line in the eIF4A-binding motif is the MIF4G/HEAT repeat domain that forms a protein–protein interaction surface. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
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Show All Figures
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Figure 5: Ded1 physically interacted with the cap-associated factors. (A) Ded1 was bound to protein A Sepharose beads crosslinked to IgG against Ded1, incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was subsequently stained with Coomassie blue. Note that Cbp80 was weakly stained, but it was clearly visible on the original gel. The ‘nonspec’ lane was a mix of all the purified proteins incubated with the resin in the absence of Ded1. (B) MalE-Cbp80 was bound to amylose resin beads (Biolabs), incubated with the indicated purified proteins, washed and the eluted proteins separated by 10% SDS PAGE. The gel was analyzed by western blot with IgG against Ded1, Cbp80 and the His6 tag of Cbp20 (Roche). Two identical gels were used to probe for Ded1 and Cbp20 because different secondary antibodies were used. Aliquots of the purified proteins (0.1 μg) are shown as markers. Note the Cbp20 protein migrated slower in the binding assay lanes due to the higher salt concentrations. (C) Purified GST-eIF4G1 fusion fragments bound to glutathione agarose beads, were incubated with Ded1, washed, eluted with reduced glutathione and separated by 10% SDS PAGE, which was subsequently stained with Coomassie blue. *The exception was the eIF4G-His6 fragment 387–952 that was retained by Ded1 bound to IgG-protein A Sepharose beads as in panel (A). Note that the 882–952 fragment gave a weak signal that was clearly visible on the original gel. For unknown reasons, the 1–387-GST fragment migrated as an aberrantly large protein. (D) The different fragments of eIF4G1 that were used in these experiments that show the locations of the previously identified binding motifs. RNA1–3 motifs bind the mRNA; motifs Pab1, eIF4E and eIF4A form direct protein–protein interactions with the corresponding proteins; and the solid line in the eIF4A-binding motif is the MIF4G/HEAT repeat domain that forms a protein–protein interaction surface. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
Mentions: The preceding experiments demonstrated direct physical interactions between Ded1 and cap-binding proteins. However, it was possible that some factors were indirectly associated with Ded1 through their interactions with the other proteins. Therefore, we individually tested various purified proteins for their ability to be retained by Ded1 fixed to Ded1-IgG Sepharose beads (Figure 5A). As a control, we incubated a mixture of the proteins with the beads in the absence of Ded1. Only Gle1 and Nab2 showed a weak nonspecific binding. The beads retained all of the indicated cap-associated factors in the presence of Ded1 except eIF4A. Both Pab1 and Cbp80 showed faint signals, but their presence was verified by western blot analysis. Ded1 bound to the beads also retained Gle1. This was consistent with previous observations that Gle1 physically and genetically interacts with Ded1 (75). As a control, we used the sec63 domain of Brr2 that is associated with protein–protein interactions (80); no interactions were detected, which indicated that only specific proteins were retained by Ded1. To verify the interactions between Ded1 and Cbp80, we bound a fusion construct between Mal-E and Cbp80 on amylose beads and tested for the ability to retain Ded1. Both Ded1 and Cbp20 were detected by western blot analysis in fractions containing MalE-Cbp80, but not in those lacking the fusion protein (Figure 5B). The Mal-E protein bound alone to the beads was unable to retain the proteins, which demonstrated that the interactions were mediated through Cbp80 (data not shown).

Bottom Line: In addition, we show that Ded1 is genetically linked to these factors.Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes.We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.

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Related in: MedlinePlus