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
Mentions: Our experiments demonstrate that Ded1 is a physical and functional component of the nuclear and cytoplasmic cap-binding complexes. We find that Ded1 is genetically linked to various CBC and eIF4F proteins, and that it physically interacts with these proteins in vitro (Figure 7). Moreover, we find that these proteins enhance the RNA-dependent ATPase activity of Ded1 in vitro. The Ded1-GFP protein actively shuttles between the nucleus and cytoplasm using both the Crm1- and Mex67-dependent nuclear export pathways, which is largely independent of the enzymatic activity of Ded1. Thus, Ded1 is probably a component of RNPs located both in the nucleus and the cytoplasm. Our mass spectrometry analyses indicate that Ded1 is associated with a large number of other proteins in cell extracts and thus in multiple different RNPs. Finally, sucrose gradients reveal that Ded1 cosediments with known cap-associated factors but that the peak concentrations of the factors do not coincide, which indicates that Ded1 interacts with a subset of these factors that form larger complexes. This distribution is not profoundly affected with rapamycin treatment, which indicates that the majority of the RNPs associated with Ded1 are not actively being translated.
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.