Abstract
The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. 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.
Highlights
The DEAD-box proteins are an ubiquitous family of proteins that are found in most organisms in all three kingdoms of life
We previously showed that Ded1 and other yeast DEAD-box proteins inhibit growth when highly expressed [4]
Our experiments demonstrate that Ded1 is a physical and functional component of the nuclear and cytoplasmic capbinding complexes
Summary
The DEAD-box proteins are an ubiquitous family of proteins that are found in most organisms in all three kingdoms of life They are implicated in all processes involving RNA, including transcription, splicing, ribosomal biogenesis, RNA export, translation and RNA decay [reviewed by [1,2,3]]. Individual proteins are distinguished by the presence of nonconserved amino- and carboxyl-terminal extensions of highly variable length and by insertions and deletions within the conserved core [4]. The amino- and carboxyl-terminal extensions, as well as variability in the conserved motifs, enable researchers to classify the proteins into different subfamilies. Motif III links the cooperative binding of the ATP and RNA ligands, which subsequently promotes the ATPase activity of the protein in association with motifs II and VI [5]. Most DEAD-box proteins lack directionality, and subsequently they are not processive
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