Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates
2003; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.1093/emboj/cdg249
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle1 June 2003free access Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates Christopher R. Trotta Christopher R. Trotta Present address: PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ, 07080 USA Search for more papers by this author Elsebet Lund Elsebet Lund Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Lawrence Kahan Lawrence Kahan Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Arlen W. Johnson Arlen W. Johnson Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, 78712 USA Search for more papers by this author James E. Dahlberg Corresponding Author James E. Dahlberg Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Christopher R. Trotta Christopher R. Trotta Present address: PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ, 07080 USA Search for more papers by this author Elsebet Lund Elsebet Lund Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Lawrence Kahan Lawrence Kahan Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Arlen W. Johnson Arlen W. Johnson Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, 78712 USA Search for more papers by this author James E. Dahlberg Corresponding Author James E. Dahlberg Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Author Information Christopher R. Trotta2, Elsebet Lund1, Lawrence Kahan1, Arlen W. Johnson3 and James E. Dahlberg 1 1Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706 USA 2Present address: PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ, 07080 USA 3Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, 78712 USA ‡C.R.Trotta and E.Lund contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2841-2851https://doi.org/10.1093/emboj/cdg249 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran·GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1–Ran·GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast. Introduction Biogenesis of eukaryotic ribosomes occurs primarily within the nucleolus and requires numerous trans-acting factors that modify and process the primary rRNA transcript and promote ribosome assembly (Hadjiolov, 1985; Warner, 1990; Maxwell and Fournier, 1995; Olson et al., 2000; Leary and Huang, 2001; Fatica and Tollervey, 2002). In vertebrates, the mature 40S and 60S subunits contain, respectively, 18S rRNA and 28S, 5.8S and 5S rRNAs plus a total of ∼80 ribosomal proteins (r-proteins) (Wool et al., 1995). The major steps of rRNA maturation have been defined (Savino and Gerbi, 1990; Sollner-Webb et al., 1996), but little is known about how the resulting subunits are exported to the cytoplasm (Bataille et al., 1990). Active nuclear export of RNA and protein is mediated by cargo-specific export receptors that pass through the nuclear pore complexes (NPCs) embedded in the nuclear envelope (Mattaj and Englmeier, 1998; Görlich and Kutay, 1999; Macara, 2001). Most RNAs and RNPs interact only indirectly with their export receptors via bi-functional adaptor proteins that contain a cargo-binding domain and a nuclear export signal (NES), which is recognized by the export receptor (Dahlberg and Lund, 1998; Izaurralde and Adam, 1998; Nakielny and Dreyfuss, 1999). Members of the importin-β family of export receptors, such as CRM1 (Fornerod et al., 1997; Stade et al., 1997), bind their cargo (plus adaptor) only in the presence of a small G-protein complexed with GTP, Ran·GTP. After the export complex enters the cytoplasm, Ran·GTP is hydrolyzed to Ran·GDP, causing release of the cargo; the adaptors, receptors and Ran are then recycled back into the nucleus, to support continued export (Lund and Dahlberg, 2001). Recently, Nmd3p, a protein conserved between Eukarya and Archaea (Belk et al., 1999; Ho and Johnson, 1999), was shown to be essential for export of 60S subunits in Saccharomyces cerevisiae (Ho et al., 2000; Gadal et al., 2001). Although the gene's name suggests a role in nonsense-mediated decay (NMD) of mRNA, Nmd3p is unlikely to play a direct role in this pathway. Nmd3p is located predominantly in the cytoplasm, associated with free 60S subunits (Ho and Johnson, 1999), but also shuttles between the nucleus and cytoplasm. The NES and the nuclear localization signal (NLS) required for shuttling are located in a C-terminal domain of Nmd3p, which is absent from archaeal NMD3 homologs. NES-deficient mutants of Nmd3p are dominant-negative inhibitors of cell growth, blocking a late step in 60S subunit biogenesis (Belk et al., 1999; Ho et al., 2000). The nuclear export of Nmd3p is dependent on Crm1p (Xpo1p) function, and inactivation of this export receptor leads to accumulation of 60S subunits within the nucleus (Ho et al., 2000; Stage-Zimmermann et al., 2000; Gadal et al., 2001). These observations have led to the model that Nmd3p binds to newly made 60S subunits in the nucleus, thereby facilitating their export to the cytoplasm via the CRM1 pathway. Here we have asked if the NMD3 protein of higher eukaryotes acts as an export adaptor for 60S subunits in the manner proposed for Nmd3p in yeast. We identified the regions of human NMD3 (hNMD3) that are important for its intracellular localization in transfected Hela cells, and determined the effects of wild-type and transport-defective hNMD3 proteins on ribosome export from Xenopus oocyte nuclei. Our results show that nuclear export of the nascent 60S subunit is coupled to export of NMD3 in vertebrate cells, and indicate that the association of NMD3 with the pre-export 60S particles allows for recognition by the export receptor CRM1. Results Nucleo-cytoplasmic shuttling of GFP–hNMD3 The amino acid sequences of hNMD3 and yeast Nmd3p share 47% identity (Belk et al., 1999; Ho and Johnson, 1999; Figure 1). Significantly, several regions in the C-terminal domain of hNMD3 have strong sequence and position homology to the proposed nuclear transport signals of yeast Nmd3p (yNmd3p). To determine if these conserved motifs affect the localization of NMD3, we generated chimeric genes encoding green fluorescent protein (GFP) fused to various forms of hNMD3 with wild-type or mutant C-terminal domains. Figure 1.NMD3 is conserved between Eukarya and Archaea. (A) Schematic diagram showing the conserved nature of the eukaryotic NMD3 proteins. This alignment was made using MACAW. Blocks of amino acid similarities are indicated by wide boxes, and positions of identity among all four sequences are indicated by vertical bars. (B) Sequence alignment of the C-terminal domains of Homo sapiens (Hs), Drosophila melanogaster (Dm), Saccharomyces cerevisiae (Sc) and Arabidopsis thaliana (At) NMD3 proteins. Conserved (black) and similar (gray) amino acid residues are indicated. NLS and NES indicate the regions resembling a basic nuclear localization signal and a leucine-rich nuclear export signal, respectively. Accession numbers are: Hs (ADD27716), Dm (AE003423.1), At (AAD24816) and Sc (S48909). Download figure Download PowerPoint The ∼83 kDa GFP–hNMD3 chimeric protein was found in both the cytoplasm and nucleus of transiently transfected HeLa cells (Figure 2A). Because this protein is too large to enter the nucleus by diffusion, it must contain an NLS (see also below, Figure 6). Within the nucleus, GFP–hNMD3 was present in both the nucleoplasm and the nucleoli. Upon short treatment of the transfected cells with leptomycin B (LMB), an inhibitor of CRM1-mediated nuclear export (Wolff et al., 1997; Kudo et al., 1998), the nucleoplasmic and nucleolar accumulation of the protein was strongly increased (Figure 2B), indicating that GFP–hNMD3 shuttles between the nucleus and cytoplasm and that its export is mediated by CRM1. Figure 2.hNMD3 is a nucleo-cytoplasmic shuttling protein. Wild-type and NES-deficient mutant GFP–hNMD3 proteins were expressed in transiently transfected HeLa cells, and their intracellular distributions were determined by direct fluorescence microscopy after 16 h of expression (A, C and E) and after further treatment of the transfected cells with leptomycin B (3 ng/ml) (+LMB) for 1.5 h (B, D and F). Bottom: comparison of the leucine-rich regions of human and yeast NMD3 proteins with the known NESs of PKI and Rev proteins. Western blot analyses using antibodies to GFP showed that more than ∼95% of the GFP was in the chimeric protein (data not shown), so fluorescence reflects the distribution of the intact fusion protein. Download figure Download PowerPoint Sequences promoting nuclear export The sequence of amino acids 480–489 of hNMD3 resembles a leucine-rich NES (Figure 2; Fischer et al., 1995; Wen et al., 1995). Substitution of the leucine residues at positions 480, 484 and 487 with alanine (NESmut) or deletion of amino acids 480–489 (ΔNES) produced mutant proteins that accumulated in both the nucleoplasm and nucleoli, even in the absence of LMB (Figure 2C–F). Mutant proteins with single (L487A) or double (L480A, L487A) alanine substitutions also displayed decreased NES activity, with the extent of nuclear accumulation correlating with the number of leucines mutated (not shown). Thus, amino acids in the 480–489 region constitute part of the NES needed for efficient export of NMD3. To test directly if this region of hNMD3 acts as an NES, a different chimeric reporter protein was used, consisting of the C-terminal 79 amino acids of hNMD3 fused to GST [GST–hNMD3(425–503)]; we note that GST alone does not diffuse out of Xenopus laevis oocyte nuclei (Askjaer et al., 1999). Upon nuclear injection, the GST fusion protein was exported rapidly to the cytoplasm when the presumptive NES was intact (Figure 3, lanes 1–6) but not when the it was mutated (lanes 7–12) or deleted (not shown). Also, the chimeric protein did not appear in the cytoplasm when the nuclear pool of Ran·GTP was depleted (lanes 13–18) (by injection of RanT24N, an inhibitor of the nuclear Ran GTP/GDP exchange factor RCC1; Izaurralde et al., 1997), showing that the protein was exported in a Ran·GTP-dependent manner. These results are consistent with the proposal that the leucine-rich 480–489 region of hNMD3 functions as an NES for CRM1–Ran·GTP-mediated export of hNMD3. Figure 3.The NES region of hNMD3 directs Ran·GTP-dependent nuclear export of a heterologous protein in Xenopus oocytes. GST fusion proteins containing the wild-type or NESmut version of the C-terminal 79 amino acids (residues 425–503) of hNMD3 were injected into nuclei of control oocytes (top and middle panels) or oocytes pre-injected with RanT24N (bottom panel) to inhibit Ran·GTP-dependent export, and export was monitored with time. One oocyte equivalent of nuclear (N) and cytoplasmic (C) extracts was analyzed by western blotting with anti-GST antibodies. Note the shorter time course for the top (15, 30 and 60 min) versus the middle and bottom (30, 60 and 120 min) panels. GST alone does not exit the nucleus during this time course (Askjaer et al., 1999; unpublished data). Download figure Download PowerPoint Sequences affecting nuclear localization The region of hNMD3 between amino acids 405 and 422 is highly basic and resembles canonical NLSs found in other proteins (Boulikas, 1993). GFP–hNMD3 mutant proteins that lacked this entire region (Δ405–422; Figure 4A) or a portion of it (e.g. Δ412–417; Figure 4B and Supplementary table S-I available at The EMBO Journal Online), or that had alanines instead of lysines at positions 405 and 406 (K405A,K406A) (Figure 4C) did not accumulate in the nucleus. Thus, the entire basic amino acid region appears to be necessary for the steady-state nuclear localization seen with the wild-type chimeric protein. Figure 4.The NLS of hNMD3 is complex. GFP–hNMD3 fusion proteins containing the indicated mutations were expressed in HeLa cells as in Figure 2, and protein localizations were monitored without (A–F) or with further treatment with LMB (G–L). The relevant amino acid sequences of the C-terminal domain of hNMD3 are shown below. Download figure Download PowerPoint Surprisingly, several non-basic regions located adjacent to this basic domain (i.e. amino acids 396–404, 439–447, 455–464 and 469–478) were also required for nuclear accumulation. Mutant proteins lacking any one of these additional regions failed to accumulate in the nucleus (Figure 4D–F; Supplementary table S-I), showing that amino acids located throughout the C-terminal domain govern the intracellular distribution of hNMD3. In spite of their cytoplasmic localization, the mutant proteins must still shuttle between the nucleus and cytoplasm, as they accumulated in the nucleus when export was inhibited by LMB (Figure 4G–L) or mutation of the NES (Supplementay table S-I). We conclude that the NLS is complex, with several elements each contributing to the efficiency of import and hence to the nucleo-cytoplasmic distribution of the protein. Sequences affecting nucleolar localization The intranuclear localization of the mutant proteins in the presence of LMB (Figure 4G–L) or upon mutation of the NES (Supplementary table S-I) revealed that amino acids located in the 396–422 region of GFP–hNMD3 acted as a nucleolar localization signal (NoLS). Proteins lacking basic amino acids in the region 405–422 accumulated primarily in the nucleoplasm rather than in the nucleolus (Figure 4G–I). In contrast, a mutant protein containing this basic region but lacking amino acids 425–503 (i.e. both the non-basic NLS sequences and the NES) accumulated prominently in nucleoli but not in the nucleoplasm (in ∼25% of the transfected cells) or solely in the cytoplasm (Figure 5B); the low percentage of cells manifesting nuclear localization of this protein may have resulted from the absence of the non-basic components of the NLS. Surprisingly, mutation of only two basic amino acids of the NLS (K405A,K406A, Δ425–503) caused exclusion from the nucleolus and led to nucleoplasmic localization in all cells (Figure 5C; see also Figure 4I). Thus, accumulation of hNMD3 in nucleoli requires some of the basic amino acids in the 405–422 region but not amino acids in the 425–503 region (see also Figure 4J–L). Figure 5.hNMD3 contains sequences required for nucleolar localization. Wild-type and mutant GFP–hNMD3 proteins were expressed as in Figure 2, and protein localizations were monitored without (A–C) or with treatment with actinomycin D (0.04 ng/ml) for 1.5 h (+ActD) (D–F). Note that treatment with ActD causes the accumulation of GFP–hNMD3 proteins in nucleolar cap structures (arrowheads in D), provided the proteins contain sequences required for nucleolar entry. Download figure Download PowerPoint Figure 6.Export of ribosomal subunits in Xenopus oocytes requires CRM1 and Ran·GTP. (A) Outline of the major pathways of rRNA processing in Xenopus (Savino and Gerbi, 1990), including the conversion of 12S rRNA to 6S rRNA, a precursor of 5.8S rRNA, that is matured after export to the cytoplasm (our unpublished results). (B and C) Requirement for the export receptor CRM1. (B) Oocytes were labeled with [32P]GTP at 0 h, and treatment with LMB (400 ng/ml) was initiated at 6 h. The intracellular distributions of newly made rRNAs in control and LMB-treated oocytes were monitored at the indicated times by analysis of 0.5 oocyte equivalents of total nuclear (N) and cytoplasmic (C) RNAs in a 1.2% agarose gel. (C) PKI NES peptides conjugated to BSA (NES–BSA) were injected into nuclei 1 h prior to labeling with [32P]GTP for 24 h, and labeled rRNAs of control and treated oocytes were analyzed as in (A). (D) Requirement for Ran·GTP. Oocytes were labeled with [32P]GTP at 0 h, and RanT24N was injected into the cytoplasm at 24 h; rRNAs of control and treated oocytes were analyzed after 24 and 40 h of labeling, as indicated. The gel mobilities of precursor and mature rRNAs are indicated, and arrowheads show the nuclear accumulation of mature rRNAs upon inhibition of ribosomal subunit export. Download figure Download PowerPoint Accumulation in the nucleolus of both the wild-type and the NES-deficient mutant (Δ425–503) protein increased when the synthesis of rRNAs was blocked by treatment with actinomycin D (ActD) (Figure 5D and E). Thus, exit of hNMD3 from nucleoli might depend on continued production of ribosomes. ActD itself is unlikely to be directly responsible for the accumulation of GFP–hNMD3 in the nucleolus, since the mutant protein hNMD3Δ425–503,K405,K406 (see above) was excluded from nucleoli even in the presence of ActD (Figure 5F). The ActD treatment also resulted in localization of some of the GFP–hNMD3 to nucleolar cap structures (arrowheads in Figure 5D) similar to those described for other nucleolar shuttling proteins (Andersen et al., 2002). CRM1 and Ran·GTP function in the export of ribosomal subunits In yeast, export of Nmd3p and 60S ribosomal subunits is coupled (Ho et al., 2000; Gadal et al., 2001). We therefore asked if export of 60S subunits and NMD3 is coupled in metazoans. To do that, we developed a manipulatable ribosome export assay using Xenopus oocytes, which contain highly amplified, very actively transcribed rRNA genes (Savino and Gerbi, 1990). Since oocyte nuclei and cytoplasm can be separated cleanly (Lund and Paine, 1990), the intracellular distributions of newly made endogenous 28S and 18S rRNAs can be used as indicators of the export of 60S and 40S subunits, respectively. In these cells, steady-state labeling of the different forms of nuclear rRNA precursors (Figure 6A) is achieved only after several hours of incubation with [α-32P]GTP, and the nuclear maturation and export of 18S rRNA is much faster than that of 28S rRNA; this results in the preferential appearance of labeled 18S rRNA in the cytoplasm at early times (6 and 15 h) after addition of label (Figure 6B, lanes 1–4; see Dunbar and Baserga, 1998). Mature newly made rRNAs accumulated in the nuclei of oocytes treated with LMB (Figure 6A, compare lanes 5 and 6 with lanes 7 and 8), indicating that CRM1 function (see Fornerod et al., 1997) is required for export of both 60S and 40S subunits. Likewise, export of both 28S and 18S rRNAs was inhibited by nuclear injection of competitor bovine serum albumin (BSA)–NES peptide conjugates (Figure 6C) but not by control BSA–NLS peptide conjugates. Export was also inhibited by depletion of Ran·GTP (by injection of RanT24N; Figure 6D). Thus, the CRM1–Ran·GTP system is required for export of both 60S and 40S subunits in Xenopus oocytes. A role for NMD3 in the export of 60S ribosomal subunits from oocyte nuclei The requirement for CRM1 function in ribosome export is consistent with a model, developed for yeast, that 60S-associated Nmd3p provides an NES that is recognized by Crm1p during export of 60S subunits (Ho et al., 2000; Gadal et al., 2001). To test directly if NMD3 functions this way in metazoans, we monitored the effects of exogenous wild-type and mutant NMD3 proteins on ribosome metabolism in oocytes. Wild-type and several of the mutant hNMD3 proteins characterized above, but lacking GFP, were made by injection of mRNAs into oocyte cytoplasms, and protein synthesis was monitored by labeling with [35S]methionine plus cysteine (Figure 7). In all cases, the 35S-labeled hNMD3 proteins accumulated in the cytoplasm (dots at lanes 2, 4, 6, 10, 12 and 14), and their nuclear levels resembled the extents of intranuclear accumulation of the corresponding GFP chimeras observed in HeLa cells. Proteins that were present in the nucleoplasm of HeLa cells, such as wild-type hNMD3 (wt) and NES-deficient mutant proteins (Figure 2A–C) with or without an NLS (K405A,K406A,NESmut; Supplementary table S-I), accumulated prominently in oocyte nuclei (Figure 7, lanes 1, 3, 9 and 11), whereas hNMD3Δ425–503, which was largely absent from the nucleoplasm of HeLa cells (Figure 5B), was present in only low amounts in oocyte nuclei (Figure 7, lane 5). However, the small amounts of hNMD3Δ425–503 that did accumulate within nuclei appeared to be nucleolar both in HeLa cells (Figure 5B) and in oocytes (our unpublished results). The mutant protein lacking both the NLS and NoLS regions (Δ405–503) could not be detected in oocyte nuclei (lanes 13 and 14). Figure 7.Intracellular distributions of hNMD3 proteins in Xenopus oocytes. Oocytes were injected with m7G-capped mRNAs encoding the indicated wild-type and mutant hNMD3 proteins and labeled with [35S]methionine for 20–24 h; nuclear (N) and cytoplasmic (C) extracts (1 and 0.5 oocyte equivalents, respectively) were analyzed by SDS–PAGE in 8% (lanes 1–8) or 10% (lanes 9–16) gels and by autoradiography. Dots indicate the newly made exogenous hNMD3 proteins. hNMD3(Δ425–503) accumulates to a much lower level in the nucleus than hNMD3(NESmut) (see text), but nonetheless is a very effective inhibitor of 60S subunit export (compare with Figure 8A, lanes 9–12). Download figure Download PowerPoint The presence of either wild-type or mutant hNMD3 proteins in the nucleus led to changes in the pattern of processing intermediates of 28S rRNA (as monitored after the labeling of nuclear rRNA precursors had reached steady state; Figure 8A). In contrast, the metabolism of 18S rRNA was not significantly affected by the exogenous hNMD3 proteins, confirming that 18S and 28S rRNAs are processed and exported independently of one another. This independence allowed us to use 18S rRNA to normalize the extent of 28S rRNA export (see legend to Figure 8). Figure 8.NES-deficient hNMD3 proteins are dominant-negative inhibitors of 60S subunit export. (A) Analyses of large rRNAs. Lanes 1–12: oocytes were pre-labeled with [32P]GTP for ∼6 h prior to injection of m7G-capped mRNAs encoding the indicated hNMD3 proteins, and the intracellular distributions of newly made rRNAs were analyzed after 40–48 h of labeling, as in Figure 6. Lanes 13–18: oocytes were injected with hNMD3 mRNAs 2 h prior to labeling with [32P]GTP, and rRNAs were analyzed after 48 h of labeling. The molar ratios of newly made 28S to18S rRNAs within the nucleus (28S:18S in N) or cytoplasm (28S:18S in C) were determined by quantification of the 32P-labeled rRNAs by phosphorImager analyses; the cytoplasmic ratio of 28S:18S rRNAs is <1.0 in control oocytes due to the slower rate of maturation of 28S rRNA than 18S rRNA (compare with Figure 6B). (B) Analyses of small rRNAs. Lanes 1–12: total RNAs (corresponding to lanes 7–18 in A) were fractionated on 8% denaturing polyacrylamide gels for analyses of 12S and 6S rRNAs (the nuclear precursors of 5.8S rRNA; compare with Figure 6A) and the mature 5S and 5.8S rRNAs. The extra bands seen in the cytoplasm of control oocytes (lane 2) are non-specific degradation products. Download figure Download PowerPoint Expression of wild-type hNMD3 left export of 28S rRNA largely unchanged but led to increased levels of the mature 28S rRNA in the nucleus at the apparent expense of the 36S and 32S pre-rRNAs, (Figure 8A, compare lanes 1 and 2 with lanes 3 and 4). Because excess hNMD3 in the nucleus did not accelerate or retard export of 28S rRNA, NMD3 apparently is neither limiting nor inhibiting for 60S subunit export in Xenopus oocytes. The increased rate of processing of precursor 28S rRNA is likely to be an indirect effect of excess nuclear hNMD3 since association of hNMD3 with 60S subunits occurs at a late stage of the maturation process (see below). Unlike wild-type hNMD3, the NES-deficient mutant proteins (NESmut and Δ425–503) strongly inhibited export of 60S subunits, as shown by the increase in the nuclear accumulation of 28S rRNA (relative to that of 18S rRNA) and the concomitant reduction in the cytoplasmic levels of 28S rRNA (Figure 8A, lanes 7 and 8, and 9 and 10). In contrast, a mutant protein lacking both NLS and NES function (Δ405–503) affected neither 60S subunit maturation nor export (Figure 8A, lanes 13 and 14, and 17 and 18). Thus, NES-deficient hNMD3 proteins act as inhibitors of 60S subunit export, provided they gain access to the nucleus. Interestingly, mutant protein that is not expected to enter nucleoli, NESmut,K405A,K406A (Supplementay table S-I), also inhibited 60S subunit export (lanes 11 and 12), showing that entry into the nucleolus may not be necessary for inhibition. We propose that, like comparable mutants of yeast Nmd3p, NES-deficient mutants of human NMD3 protein inhibit export of 60S subunits by competing with endogenous wild-type NMD3 for a binding site on nuclear 60S subunits. As a consequence, the nascent 60S subunits are deprived of an NES-containing export adaptor. Association of hNMD3 with nascent 60S ribosomal subunits To test directly if hNMD3 binds to 60S subunits in oocytes, we monitored ribosome association of wild-type and NES-deficient forms of hNMD3 that were fused to maltose-binding protein (MBP–hNMD3). The injected MBP fusion proteins (made in bacteria) had the same nucleo-cytoplasmic distributions and the same effects on 60S subunit export as the comparable untagged hNMD3 proteins produced in oocytes (data not shown). As shown by sucrose gradient centrifugation, MBP–hNMD3 associated with 60S subunits in both nucleoplasmic and cytoplasmic extracts, but not with 40S subunits nor 80S ribosomes (Figure 9A); also, this pattern of association was not affected by the presence or absence of a functional NES on the hNMD3 (data not shown). As expected, anti-MBP antibodies specifically co-precipitated 28S rRNA but not 18S rRNA from both nucleoplasmic (Figure 9B) and cytoplasmic (not shown) extracts. The lack of pre-28S rRNAs from the immunoprecipitated ribosomes (Figure 9B) demonstrated that hNMD3 bound only to subunits that were in the late stages of maturation. Figure 9.hNMD3 associates with nascent 60S ribosomal subunit in both the nucleus and the cytoplasm. (A) Nucleoplasmic and cytoplasmic extracts of oocytes injected with MBP–hNMD3-NESmut fusion protein and labeled with [32P]GTP were fractionated on 10–40% sucrose gradients, and the distributions of newly made rRNAs (top panels) and MBP–hNMD3 (bottom panels) were determined by agarose gel electrophoresis and western blotting with anti-MBP antibodies, respectively. In the experiment shown, the nucleoplasmic extract was prepared from oocytes treated with VSV M protein (an inhibitor of nuclear export; Her et al., 1997) to increase the levels of 40S and 60S export complexes in the nucleoplasm, but both wild-type and NESmut hNMD3 proteins are also associated with nascent 60S subunits in nucleoplasmic extracts of untreated control oocytes [unpublished data; compare with (B)]. (B and C) Nuclear extracts of oocytes injected with wild-type (WT) or NESmut MBP–hNMD3 proteins were immunoprecipitated with anti-MBP antibodies, and the large (B) and small (C) rRNAs of the bound (Ppt) and unbound (Sup) fractions and the total (Tot) extract were analyzed by gel electrophoresis as in Figure 8. M: marker rRNAs as indicated. Note that the nucleoplasmic extracts are devoid of nucleoli, which contain the majority of the 32S, 36S and 40S precursor rRNA, and which are abundant in total nuclear RNAs (Figure 6B). Download figure Download PowerPoint We were unable to detect a presumptive pre-export complex containing nuclear 60S subunits, NMD3, CRM1 and Ran·GTP (data not shown). Perhaps such complexes rapidly dissociated during sample preparation upon exposure to cytoplasmic RanGAP. Similar observations have been made for yeast (Nissan et al., 2002; A.Johnson, unpublished). The nuclear 60S subunits that were precipitated by anti-MBP antibodies
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