Abstract
The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.
Highlights
The biogenesis of eukaryotic ribosomal subunits begins in the nucleolus with the generation of a common 35S precursor transcript, which is processed by a series of cleavage events into the mature 18S, 25S and 5.8S rRNAs [1]
Associated with free ribosomal subunits and might have a function on the subunits prior to translation. Considering that this RNA-helicase is necessary for the directional transport of mRNAs into the cytoplasm [12, 13], we investigated if Dbp5 would be required for the nuclear export of pre-ribosomal subunits
Fluorescence in situ hybridization (FISH) experiments with fluorescent 25S and 18S rRNA probes revealed that almost all cells of the temperature-sensitive dbp5 mutants rat8-2 and rat8-7 accumulate pre-60S and pre-40S subunits in their nuclei upon shift to their restrictive temperatures (Fig 2A and 2B; for all split channels and merged pictures see S1A and S1B Fig)
Summary
The biogenesis of eukaryotic ribosomal subunits begins in the nucleolus with the generation of a common 35S precursor transcript, which is processed by a series of cleavage events into the mature 18S, 25S and 5.8S rRNAs [1]. Various assembly factors and ribosomal proteins associate with the 35S pre-rRNA to form the 90S precursor particle that is subsequently split into a pre-60S and a pre-40S subunit. The pre-60S particle interacts with the nuclear export signal (NES)containing adaptor protein Nmd in Saccharomyces cerevisiae (human NMD3), which recruits the export receptor Xpo (CRM1/Exportin1) [2,3,4,5]. Xpo and the Ran GTPase system are involved in the export of the pre-40S subunit [7]. The well-established mRNA export receptor heterodimer Mex67-Mtr (TAP-p15), which directly contacts the rRNA, is necessary for the transport of both pre-ribosomal particles [9, 10]. Upon passage through the NPC, further maturation steps including the final processing of the 20S pre-rRNA to the mature 18S rRNA occur in the cytoplasm, before the ribosomal subunits are competent for translation initiation [11]
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