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