Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

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.

Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

“…Yeast cells expressing green fluorescent protein (GFP)-tagged Rps2p were cultivated in 150 ml YNB medium to mid-log phase and harvested by centrifugation at 1,650 ϫ g for 4 min at 4°C. GFP trap assays were performed as described by Neumann et al (55). Cells were washed with phosphatebuffered saline (PBS; 8 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 150 mM NaCl) and subsequently lysed with glass beads in the presence of 250 l breaking buffer (PBS, 3 mM KCl, 2.5 mM MgCl 2 , 0.5% Triton X-100, 1 tablet of cOmplete EDTA-free protease inhibitor [Sigma-Aldrich] per 50 ml, and phosphatase inhibitors as described above).…”

Quick or quality? How mRNA escapes nuclear quality control during stress

“…The proper coverage of large RNA-containing molecules with export receptors is also required for the export of ribosomal subunits, as missing export factors result in the accumulation of subunits in the nucleus. [40][41][42][43][44][45][46] The last processing step that results in decoration of the mRNA with the guard proteins is the formation of the 3 0 end and synthesis of the poly(A) tail. Here the poly(A)-binding protein Nab2, together with its mainly cytoplasmic homolog Pab1, 47,48 controls length and quality of the 3 0 taila process that is antagonized by the exosome component Rrp6.…”

Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

“…To date, 21 RNA helicases have been suggested to act during the process of ribosome biogenesis in yeast with eight implicated in SSU maturation, ten in LSU biogenesis and three in the assembly of both subunits [20]. A wealth of research has provided information on the binding sites and potential functions of many of these RNA helicases [21][22][23][24][25][26][27][28][29][30][31][32][33], with several RNA helicases implicated in promoting the release of specific snoRNPs from early pre-ribosomal particles [21,22,26,27,30,31] and others suggested to catalyse pre-rRNA rearrangements that induce formation of mature rRNA conformations facilitating the recruitment of RPs and/or AFs [23,32,34]. However, in the case of other RNA helicases that have been found associated with pre-ribosomal particles, little is known about the timing of their association, the interactions they make within pre-ribosomes and what contribution their activity makes to the assembly process.…”