Abstract
We have characterized a novel mRNA-binding protein, designated hrp84, in the dipteran Chironomus tentans and identified it as a DEAD-box RNA helicase. The protein contains the typical helicase core domain, a glycine-rich C-terminal part and a putative nuclear export signal in the N terminus. The protein belongs to the Ded1 subgroup of DEAD-box helicases, which is highly conserved from yeast (Ded1p) to mammals (DDX3). In tissue culture cells, hrp84 is present both in the nucleus and cytoplasm and, as shown by in vivo UV cross-linking, is bound to mRNA in both compartments. Immunoprecipitation experiments revealed that hpr84 is associated with the C. tentans homologue (ctYB-1) of the vertebrate Y-box protein YB-1 both in the nucleus and cytoplasm, and the two proteins also appear together in polysomes. The interaction is likely to be direct as shown by in vitro binding of purified components. We conclude that the mRNA-bound hrp84.ctYB-1 complex is formed in the nucleus and is translocated with mRNA into the cytoplasm and further into polysomes. As both Ded1 and YB-1 are known to regulate the initiation of translation, we propose that the RNA helicase-Y-box protein complex affects the efficiency of mRNA translation, presumably by modulating the conformation of the mRNP template.
Highlights
Despite extensive studies, the precise mechanism of action of the DEAD-box proteins is still unclear
Another example is the interplay in mammalian cells between the eukaryotic translation initiation factors eIF4A and eIF4B. eIF4A is an RNA helicase that unwinds secondary structures in the 5Ј-untranslated region (5Ј-UTR) of the mRNA, which enables the small ribosomal subunit to bind to the cap region of the mRNA and scan along the RNA to reach the start codon AUG [17]
We have identified a novel RNA helicase in C. tentans, which belongs to the Ded1 subfamily of DEAD-box helicases, known to be involved in translation initiation
Summary
The precise mechanism of action of the DEAD-box proteins is still unclear. The information is still limited in both Xenopus and Drosophila, it is close at hand to suggest that the RNA helicase promotes the binding of the Y-box protein to the mRNA and in this way blocks translation. One of the monoclonal antibodies, designated mAb 4E11, recognized an 84-kDa protein in the RNP extract from tissue culture cells as shown by Western blot analysis (Fig. 1A).
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