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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-IgG pull-down experiments. Yeast extracts were incubated with Ded1-IgG-Sepharose beads, washed, the proteins eluted and then the recovered proteins were separated by an 8% SDS polyacrylamide gel electrophoresis (PAGE) for Cbp80 and eIF4G1, and by an 10% SDS PAGE for the others. IgG, rabbit serum after induction with injected, purified, Ded1 protein; Pre, rabbit pre-immune serum; Ext, the equivalent of 1 μl of yeast extract. Note that for each set of experiments, the material was analyzed on the same gel with the same extract; an empty lane was placed between the pull-down lanes and the extract lanes to avoid potential cross-contamination. The separated proteins were transferred to membranes, cut into strips and probed with antibodies against the indicated proteins. Cbp20 was expressed from a plasmid incorporating an amino-terminal HA tag, and it was detected with IgG against HA. The other proteins were detected from the wildtype BY4742 strain with antibodies specific to each protein. The molecule weight markers were PAGE Ruler from Thermos Scientific.
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Figure 3: Ded1-IgG pull-down experiments. Yeast extracts were incubated with Ded1-IgG-Sepharose beads, washed, the proteins eluted and then the recovered proteins were separated by an 8% SDS polyacrylamide gel electrophoresis (PAGE) for Cbp80 and eIF4G1, and by an 10% SDS PAGE for the others. IgG, rabbit serum after induction with injected, purified, Ded1 protein; Pre, rabbit pre-immune serum; Ext, the equivalent of 1 μl of yeast extract. Note that for each set of experiments, the material was analyzed on the same gel with the same extract; an empty lane was placed between the pull-down lanes and the extract lanes to avoid potential cross-contamination. The separated proteins were transferred to membranes, cut into strips and probed with antibodies against the indicated proteins. Cbp20 was expressed from a plasmid incorporating an amino-terminal HA tag, and it was detected with IgG against HA. The other proteins were detected from the wildtype BY4742 strain with antibodies specific to each protein. The molecule weight markers were PAGE Ruler from Thermos Scientific.

Mentions: We next asked what were the factors that were physically associated with Ded1 in the cell. We initially tried the double-affinity purification procedure that uses Ded1 fused to the protein A and the calmodulin-binding proteins to isolate complexes containing Ded1 from cell extracts [TAP-tag method; (73)]. However, the long loading times on the columns and poor recovery of Ded1 precluded reliable identification of the cofactors. Nevertheless, the results from the MALDI-TOF MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) analyses were consistent with Ded1 interacting with the translational machinery (data not shown). Therefore, we used IgG against Ded1, attached to protein A-Sepharose beads, to pull down the complexes in yeast extracts. We tried various conditions and found that light digestion of the extracts with RNase A gave the best yields and quality of material. Western blot analyses of the separated proteins revealed the expected proteins eIF4E, eIF4G1, Pab1, Cbp20, Cbp80 and Nab2 (Figure 3). We also recovered a weak signal for eIF4G2 in some experiments. Ded1 was previously found to interact with Dhh1 and Gle1 (74,75), and we were able to identify these proteins as well (Figure 3). Moreover, we recovered eIF4A. Thus, Ded1 was associated with both nuclear and cytoplasmic proteins that are known components of mRNA RNPs. These results were verified by nanoLC-MS/MS analysis of the polyacrylamide gels (Supplemental Tables S1 and S2).


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-IgG pull-down experiments. Yeast extracts were incubated with Ded1-IgG-Sepharose beads, washed, the proteins eluted and then the recovered proteins were separated by an 8% SDS polyacrylamide gel electrophoresis (PAGE) for Cbp80 and eIF4G1, and by an 10% SDS PAGE for the others. IgG, rabbit serum after induction with injected, purified, Ded1 protein; Pre, rabbit pre-immune serum; Ext, the equivalent of 1 μl of yeast extract. Note that for each set of experiments, the material was analyzed on the same gel with the same extract; an empty lane was placed between the pull-down lanes and the extract lanes to avoid potential cross-contamination. The separated proteins were transferred to membranes, cut into strips and probed with antibodies against the indicated proteins. Cbp20 was expressed from a plasmid incorporating an amino-terminal HA tag, and it was detected with IgG against HA. The other proteins were detected from the wildtype BY4742 strain with antibodies specific to each protein. The molecule weight markers were PAGE Ruler from Thermos Scientific.
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Related In: Results  -  Collection

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Figure 3: Ded1-IgG pull-down experiments. Yeast extracts were incubated with Ded1-IgG-Sepharose beads, washed, the proteins eluted and then the recovered proteins were separated by an 8% SDS polyacrylamide gel electrophoresis (PAGE) for Cbp80 and eIF4G1, and by an 10% SDS PAGE for the others. IgG, rabbit serum after induction with injected, purified, Ded1 protein; Pre, rabbit pre-immune serum; Ext, the equivalent of 1 μl of yeast extract. Note that for each set of experiments, the material was analyzed on the same gel with the same extract; an empty lane was placed between the pull-down lanes and the extract lanes to avoid potential cross-contamination. The separated proteins were transferred to membranes, cut into strips and probed with antibodies against the indicated proteins. Cbp20 was expressed from a plasmid incorporating an amino-terminal HA tag, and it was detected with IgG against HA. The other proteins were detected from the wildtype BY4742 strain with antibodies specific to each protein. The molecule weight markers were PAGE Ruler from Thermos Scientific.
Mentions: We next asked what were the factors that were physically associated with Ded1 in the cell. We initially tried the double-affinity purification procedure that uses Ded1 fused to the protein A and the calmodulin-binding proteins to isolate complexes containing Ded1 from cell extracts [TAP-tag method; (73)]. However, the long loading times on the columns and poor recovery of Ded1 precluded reliable identification of the cofactors. Nevertheless, the results from the MALDI-TOF MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) analyses were consistent with Ded1 interacting with the translational machinery (data not shown). Therefore, we used IgG against Ded1, attached to protein A-Sepharose beads, to pull down the complexes in yeast extracts. We tried various conditions and found that light digestion of the extracts with RNase A gave the best yields and quality of material. Western blot analyses of the separated proteins revealed the expected proteins eIF4E, eIF4G1, Pab1, Cbp20, Cbp80 and Nab2 (Figure 3). We also recovered a weak signal for eIF4G2 in some experiments. Ded1 was previously found to interact with Dhh1 and Gle1 (74,75), and we were able to identify these proteins as well (Figure 3). Moreover, we recovered eIF4A. Thus, Ded1 was associated with both nuclear and cytoplasmic proteins that are known components of mRNA RNPs. These results were verified by nanoLC-MS/MS analysis of the polyacrylamide gels (Supplemental Tables S1 and S2).

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