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 indicated that Ded1 was in complexes containing the identified proteins, but they did not demonstrate a direct or functional interaction between the proteins. However, it seemed likely that Ded1 was part of the nuclear and cytoplasmic cap-binding complexes. To test this, we purified the different recombinant proteins from E. coli (Supplemental Figure S5) and tested their ability to form complexes on 7-methyl-GTP Sepharose beads. Both Cbp20 and eIF4E have high affinity for 7-methylguanosine. We bound these proteins to the beads, added various combinations of the purified proteins and then eluted the proteins retained (Figure 4). To facilitate the analyses, we used a shorten version of eIF4G1 (eIF4G9; amino acids 160–492) that was known to retain its capacity to interact with Pab1 and eIF4E (43). Of the proteins tested, only the polyadenosine-binding protein Nab2 showed a weak, nonspecific affinity for the beads.
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.