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First published online 30 April 2003
doi: 10.1242/jcs.00464

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Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway

Swiss Federal Institute of Technology (ETH) Zürich, Institute of Biochemistry, HPM F11.1, CH-8093 Zürich, Switzerland

View larger version (40K): [in a new window]   Fig. 1. Leptomycin B (LMB) affects the intracellular localization of rpS5-EGFP and rpL29-EGFP. (A) Localization of rpS5-EGFP and rpL29-EGFP at different times after transfection. HeLa cells were grown on coverslips and transiently transfected with constructs coding for EGFP fusions of rpS5 or rpL29. Cells were fixed with 3.7% paraformaldehyde and further processed for analysis by fluorescence microscopy at 16 hours, 25 hours and 38 hours after transfection, respectively. (B) LMB-sensitive localization of rpS5-EGFP and rpL29-EGFP. HeLa cells were transiently transfected with constructs coding for EGFP fusions of rpS5 and rpL29, respectively. After 16 hours, cells were transferred into fresh medium with or without LMB (10 ng/ml). Five or 18 hours later, cells were fixed and analyzed by fluorescence microscopy.

View larger version (108K): [in a new window]   Fig. 2. Leptomycin B (LMB)-sensitive localization of endogenous rpS6 and rpL23a. (A) Specificity of the anti-L23a and anti-S6 antibodies. Total HeLa cell proteins were separated by 8% SDS-PAGE followed by immunoblotting using anti-L23a and anti-S6 antibodies. Note that both antibodies are highly specific and detect a single protein band. (B) HeLa cells were either left untreated or treated with LMB for 18 hours. Then, cells were fixed/permeabilized with either 3.7% paraformaldehyde (PFA)/acetone or with acetone/methanol. The localization of rpL23a or rpS6 was analyzed by indirect immunofluorescence using anti-L23a and anti-S6 antibodies, respectively. Images were taken by confocal fluorescence microscopy.

View larger version (118K): [in a new window]   Fig. 3. Nuclear accumulation of rRNAs upon leptomycin B (LMB) treatment of HeLa cells. Localization of rRNAs was analyzed in untreated HeLa cells or cells that had been treated with LMB for 18 hours by fluorescent in situ hybridization (FISH) using digoxigenin-labeled oligonucleotides directed to 18S and 28S rRNA, respectively. To control for the specificity of the detection, cells were either treated with RNaseA before FISH analysis or the probes were omitted (upper panels). Images were taken by confocal fluorescence microscopy.

View larger version (51K): [in a new window]   Fig. 4. Nuclear export of ribosomal subunits is competed by NES peptides. (A) Scheme illustrating the processing of pre-rRNA along two co-existing pathways in Xenopus oocytes (modified according to Peculis, 1997) (Peculis, 1997). Cleavages occur in an orderly fashion at numerous sites indicated by arrows. This leads to the removal of externally and internally transcribed spacers (ETS, ITS) to yield mature 5.8S, 18S and 28S rRNA. (B) Competition of the CRM1-mediated NES export pathway interferes with nuclear exit of both ribosomal subunits. Xenopus oocytes were injected with [-32P]GTP. After 3 or 5 hours, PKI-NES wild-type or mutant peptides conjugated to BSA were injected into the nucleus. At the indicated times, RNA was isolated from nuclear (N) or cytoplasmic (C) fractions, separated by agarose gel electrophoresis and analyzed first by phosphoimaging for quantification and thereafter by autoradiography.

View larger version (36K): [in a new window]   Fig. 5. hNMD3 possesses a conserved, C-terminal NES sequence. (A) Scheme illustrating the position of sequence motifs in NMD3 from different species and constructs used for transient expression. NMD3 proteins from all species possess an N-terminal conserved domain containing 4 putative zinc fingers (gray boxes). NMD3s in eukaryotes have acquired a C-terminal domain harboring the NLS (white box) and two potential C-terminal NES sequences, according to Ho et al. and Gadal et al. (Ho et al., 2000b; Gadal et al., 2001). Deletion of NES1 (black box) in S.c.Nmd3p dramatically affects cell growth and leads to a nuclear localization of the protein, whereas the potential NES2 (striped box) does show some deviation from the NES consensus sequence but in conjunction with NES2 is needed for viability of yeast (Gadal et al., 2001). The NES1 sequence is conserved throughout higher eukaryotes. Conserved residues are highlighted in bold. For comparison, the PKI NES is shown. Accession numbers of the different NMD3 proteins are the following: S.c. P38861, S.p. Q09817, H.s. NP_057022, M.m. NP_598548, A.g. EAA11292, D.m. CAB42049, C.e. CAA96689, A.t. AAL07089 and O.s. AAK00432. Full-length wild-type hNMD3, C-terminal deletion mutants lacking NES1 (hNMD3C27), or both NESs (hNMD3C71), and an NES mutant (hNMD3-NESmut), in which L487 and I489 were changed to alanines, are depicted. (B) hNMD3 contains an NES sequence in the last 20 amino acids. GFP fusions of the different constructs as presented in A were expressed in transiently transfected HeLa cells. Eighteen hours post-transfection, the cells were transferred into fresh medium without or with leptomycin B (LMB) and fixed after 4 hours. The intracellular distribution of the individual proteins was determined by fluorescence microscopy.

View larger version (57K): [in a new window]   Fig. 6. hNMD3 binds to 60S subunits in vitro. (A) Saturable binding of recombinant hNMD3 to 60S ribosomal subunits. Twenty-five pmol of purified HeLa cell 60S subunits (lanes 3 to 12) were incubated with increasing amounts of 6His-hNMD3 (10, 20, 40, 60 or 80 pmol). Ribosomal subunits and associated hNMD3 were pelleted by sedimentation. The proteins in the pellet (P) and supernatant (S) were separated by 10% SDS-PAGE followed by Coomassie blue staining. Load of proteins in the unbound fractions (supernatants) equals the load of the ribosomal pellet fractions. Note that there is no sedimentation of 6His-hNMD3 in the absence of 60S subunits (lanes 1 and 2). (B) hNMD3 cosediments with 60S subunits. A mixture of each 25 pmol purified 40S and 60S subunits was incubated with 60 pmol of 6His-hNMD3 and then fractionated on a 10-40% sucrose density gradient. Fractions from the gradient were analyzed by Western blotting using antibodies directed to rpL10 and rpS6 as markers for the migration of 60S and 40S subunits, respectively. Detection of hNMD3 was by an antibody that recognizes the N-terminal 6His-tag of recombinant hNMD3. Note that hNMD3 comigrates with 60S subunits and that, in addition, it is found on the top of the gradient as free protein but not in association with 40S subunits.

View larger version (17K): [in a new window]   Fig. 7. hNMD3 can engage into a complex with CRM1/RanGTP. (A) RanGTP stimulates the direct binding of CRM1 to NMD3. Recombinant 2z-hNMD3 FL (lanes 2 to 4) or 2z-hNMD3C27 (lanes 5 to 7) were incubated with recombinant CRM1 in the absence or presence of RanQ69L(GTP). 2z-hNMD3 associated factors were retrieved from the reaction mixtures by IgG-Sepharose. Bound proteins were separated by SDS-PAGE and detected by Coomassie blue staining. The amount of unspecific binding of CRM1 in the presence of RanGTP to IgG-Sepharose was also determined (lane 1). (B) 2z-hNMD3 forces CRM1 into a complex with RanGTP. Apparent dissociation constants of complexes between RanGTP and CRM1 were estimated in the presence of either 2 µM 2z-hNMD3, 2z-hNMD3C27 or 6His-hNMD3 using the RanGTPase assay. Note that only 2z-hNMD3 and only to some extent 6His-hNMD3, but not 2z-hNMD3C27, are able to force CRM1 into complex with Ran[-32P]GTP. The complex formation of RanGTP and CRM1 protects the GTP on Ran from RanGAP-induced GTP hydrolysis.