The transport of proteins and RNAs in both directions across the nuclear envelope constitutes a set of processes called nucleocytoplasmic trafficking(1, 2) . Both the import of proteins and the export of RNAs occur via the nuclear pore complex. However, the steps responsible for the delivery of substrates to the pore complex, variously referred to as ``targeting'' and ``docking,'' differ for protein import and RNA export. For many proteins, the nuclear localization signals (NLSs) ()that direct the targeting phase of import have been delineated(3) , and recently, a number of factors thought to mediate import have been identified using cell-free fractionation and reconstitution assays(4, 5, 6, 7, 8) . Protein import can also be negatively controlled by cytoplasmic retention or NLS masking(1) . While most nuclear proteins are imported by a single predominant targeting pathway(9) , certain U small nuclear ribonucleoprotein particles are targeted to the pore complex by a distinct mechanism(10, 11) .

The export mechanisms for tRNA(12) , mRNA(13, 14) , signal recognition particle RNA(15) , rRNA (ribosomal subunits)(15, 16) , and snRNA (14, 18, 19) are saturable and, hence, are receptor-mediated. In most cases, with the exception of rRNA, which has not been investigated, and tRNA, which appears to have general competitive effects (see below), the saturable step in the export of each RNA is mediated by specific factors. Mutagenesis studies indicate that the cis-acting signals that direct the export of tRNA(12, 20) , signal recognition particle RNA(15) , snRNAs(18, 21, 22) , 5 S RNA(23) , and mRNA(15, 24) are complex and may involve recognition of RNA secondary structure, covalent modifications, and associations with RNA-binding proteins. Although the 5`-mGpppN cap of RNA polymerase II transcripts does not appear to be essential for export(15, 18) , it probably does serve as a key enhancer of export. Thus, the presence of a cap increases mRNA and U snRNA export rates, and free cap dinucleotide inhibits the export of capped RNAs (reviewed in (21) , but see (18) ).

Beginning with their sequestration within transcriptional and post-transcriptional processing and ribonucleoprotein assembling centers, the transient retention of RNAs within the nucleus is a controlling factor in both the constitutive and regulated export of RNAs(25, 26, 27, 28, 29) . RNA-specific nuclear retention mechanisms and virus-induced export blocks also occur (reviewed in (29) ). For example, the retention of HLS-DRA mRNA was recently shown to be determined by a signal located in the mRNA's 3`-untranslated region (30) . Fabre et al.(31) recently discovered a class of pore complex proteins in yeast that bind RNA and could function as general RNA docking sites during the latter stages of RNA targeting to the pore complex.

In this study, we show that a saturable factor in 40 S ribosomal subunit export is specific and is not limiting for tRNA export. Also, we present evidence that the rate-limiting step for tRNA export is the dissociation from high affinity retention sites that can be titrated with tRNA, mRNA, and rRNA.

MATERIALS AND METHODS

Preparation of Substrates

Yeast tRNA (Sigma) was purified by phenol:chloroform and chloroform extractions followed by several ethanol precipitations and was suspended in water. Alternatively, tRNA was purified by extraction from 8 M urea, 10% polyacrylamide gels.

P-Labeled ribosomes were purified from Tetrahymena thermophila grown overnight on phosphate-depleted medium supplemented with PO(33) . Cells were pelleted at 5000 g for 1 min and resuspended in 30 mM Tris, pH 7.5, 50 mM KCl, 10 mM MgCl, 0.5% Nonidet P-40, 2 mM dithiothreitol, 10 µg/ml heparin, and 1 µg/ml each aprotinin, leupeptin, and pepstatin. Cells were vortexed and incubated on ice for 15 min. The homogenate was spun for 10 min to pellet cell debris, and the resulting supernatant was layered over 1 M sucrose, 50 mM Tris, pH 7.5, 500 mM KCl, 10 mM MgCl and centrifuged in a TLA100.2 rotor at 4 °C for 36 min at 100,000 rpm in a Beckman TL-100 ultracentrifuge. The ribosome pellet was resuspended in dissociation buffer (50 mM Tris, 500 mM KCl, 1 mM MgCl), centrifuged through 10-40% sucrose gradients prepared in the same buffer, and fractionated using a Searle Densi-Flo IIC fractionator linked to an Isco UA5 absorbance monitor. Fractions containing 40 S and 60 S subunits were pooled, and the subunits were pelleted and stored at -80 °C. Subunit pellets were resuspended in oocyte injection buffer (10) containing 2 mM MgCl. RNA extracted from the 40 S subunits and analyzed on a 1.0% formaldehyde-agarose gel reveals one band, corresponding to the 18 S RNA (data not shown). Unlabeled rRNA for use in competition experiments was purified from 40 S or 60 S ribosomal subunits or from polysomes by phenol:chloroform extraction and ethanol precipitation.

Microinjections and Analysis of Export

Oocytes were fixed in 10% trichloroacetic acid, enucleated, and solubilized in NCS-II (Amersham Corp). Scintillation fluid and glacial acetic acid were added according to the manufacturer's guidelines, and values for percent export were determined by scintillation counting. Intranuclear concentrations of injected materials were calculated assuming a nuclear volume of 40 nl.

To verify that export substrates were exiting the nucleus as intact RNAs, oocytes were injected with [P]tRNA or P-labeled 40 S subunits and incubated for 30 min. Injected oocytes were fixed in 50 mM sodium acetate, pH 5.2, and enucleated, and the RNA was extracted from nuclear and cytoplasmic fractions as described(10) . Total nuclear and cytoplasmic RNAs were analyzed on 8 M urea, 10% polyacrylamide gels or on 0.8% formaldehyde-agarose gels, followed by autoradiography.

RESULTS

The kinetics of tRNA (12) and ribosomal subunit (16, 17) export from Xenopus oocyte nuclei have been characterized and shown to be saturable. To ensure that we had purified export-competent substrates, we first verified that the export of tRNA and ribosomal subunits was saturable by performing a simple self-competition analysis. In vitro transcribed [P]tRNA was injected into Xenopus oocyte nuclei, and export was assayed by monitoring the appearance of [P]tRNA in the cytoplasm. Microinjected tRNAs were stable for the time course of these experiments as indicated by the observations that the total counts/minute/oocyte remained relatively constant over the time course of these experiments and the percent export values obtained by the scintillation counting of whole cytoplasms and nuclei (see ``Materials and Methods'') agreed with those obtained by excising acrylamide gel slices containing the nuclear and cytoplasmic [P]tRNAs. We, like others(13) , found that nuclear microinjection leads to initial leakage of 10-20% of the injected tRNA into the cytoplasm. This phenomenon was peculiar to tRNA as we did not observe leakage of P-labeled ribosomal subunits, I-bovine serum albumin, or colloidal gold (colored red) following their microinjection.

When tRNA was injected to an intranuclear concentration of 6 nM, export proceeded as shown in Fig. 1A, with a t of 30 min (see also (12) ). Self-competition was demonstrated by adding unlabeled yeast tRNA to the injection solution to a final concentration of 60 µM. This competed the percent export of [P]tRNA to slightly above background levels (20%).

Figure 1: The export of tRNA and ribosomal subunits is saturable. The results are representative of three experiments in which at least 10 oocytes were analyzed for each time point. Errorbars here and on the other figures indicate standard deviation and are sometimes too small to visualize on these graphs. A, in vitro transcribed [P]tRNA (6 nM) was injected into the nuclei of Xenopus oocytes, and the time course of export was assayed in the absence () or presence (⧫) of 60 µM unlabeled tRNA. B, P-labeled 40 S subunits purified from Tetrahymena were injected into oocyte nuclei at a concentration of 0.07 () or 0.7 (⧫) µM, and export was assayed at various times after injection. Preparation of export substrates is described under ``Materials and Methods.''

Analogous results were obtained for the export of P-labeled ribosomal subunits (Fig. 1B). P-Labeled subunits were purified from T. thermophila grown in the presence of PO. Tetrahymena is a convenient source of ribosomes, and Tetrahymena ribosomes had previously been shown to be exported from Xenopus oocyte nuclei(17) . P-Labeled 40 S ribosomal subunits (0.07 µM) were exported with a t of between 15 and 30 min. The injection of a 10-fold higher concentration of P-labeled 40 S subunits resulted in a significantly lower percent export rate, indicative of saturation kinetics (Fig. 1B). As previously shown by others(16, 17) , assessment by gel electrophoresis and autoradiography confirmed that the P-labeled 18 S rRNA of microinjected 40 S subunits remained full length in both the nucleus and cytoplasm during the course of these experiments (data not shown).

To prepare for competition studies between tRNA and ribosomal subunits, we determined the concentrations required for maximal tRNA and ribosomal subunit self-competition. The concentration of tRNA needed to achieve maximal inhibition of [P]tRNA export (10 µM; see Fig. 5A) was roughly 20 times higher than the concentration of ribosomal subunits needed to achieve maximal inhibition of P-labeled ribosomal subunit export (0.5 µM; data not shown). These concentrations are in accord with previously published results(12, 13, 14, 16, 17) .

Figure 5: The export of tRNA is concentration-dependent. A, [P]tRNA was microinjected into oocyte nuclei with increasing concentrations of unlabeled tRNA. Percent export was determined after 20-min incubations. Injection solutions contained 6 nM [P]tRNA and sufficient unlabeled tRNA to make up the various concentrations. B, shown is the dose dependence of tRNA export in the presence of rRNA. Injections were performed as described for A, except that 1.6 µM rRNA was included in each injection solution.

tRNA Export Is Enhanced by Ribosomal Subunits or rRNA

Figure 2: tRNA export is stimulated by co-injection with 40 S ribosomal subunits or purified rRNA. A, in vitro transcribed [P]tRNA (6 nM) was injected into nuclei of Xenopus laevis oocytes in the absence () or presence (⧫) of 40 S subunits (0.7 µM), and export was assayed as described under ``Materials and Methods.'' B, [P]tRNA (6 nM) was microinjected into nuclei in the absence () or presence (⧫) of purified ribosomal RNA (1.6 µM).

The reciprocal experiment was performed to test the effect of saturating concentrations of tRNA on P-labeled 40 S subunit export. As shown in Fig. 3, 1.6 µM rRNA had a large inhibitory effect on P-labeled 40 S subunit export that is consistent with competition. Although tRNA export was not inhibited by saturating concentrations of 40 S subunits or rRNA (Fig. 2), the export of P-labeled 40 S subunits was partially inhibited by concentrations of tRNA (60 µM) that effectively compete [P]tRNA export. This argues that the partial inhibition of P-labeled 40 S subunit export is not due to competition for the same factor that is limiting for tRNA export. Rather, this weak effect may be indirect, artifactual, or indicative of another shared intermediate. Partial inhibition of tRNA and mRNA export by similar high concentrations of homopolymeric competitor RNAs was observed by Jarmolowski et al.(14) .

Figure 3: Effects of tRNA on 40 S subunit export. The export of 0.07 µMP-labeled 40 S subunits was assayed in the absence () or presence of tRNA (⧫) or rRNA (&cjs3409;). Export was assayed as described under ``Materials and Methods.''

Stimulation of tRNA Export by Ribosomal Subunits Depends on the Intranuclear Concentration of Subunits and Not on Their Export Flux

Figure 4: [P]tRNA export as a function of 40 S subunit concentration. The export of microinjected [P]tRNA (6 nM) was assayed in the presence of increasing concentrations of unlabeled 40 S subunits. In each case, injected oocytes were incubated for 20 min. Maximal inhibition of ribosomal subunit export occurs at 0.5 µM.

tRNA Export Rate Is Limited by Titratable Retention Sites

The observation that tRNA stimulates its own export does not address whether tRNA and rRNA stimulate export by the same mechanism. To investigate whether the stimulation of tRNA export by rRNA and tRNA is the result of titrating the same or different retention sites, we repeated the tRNA dose-dependence experiment in the presence of saturating concentrations of rRNA. As shown in Fig. 5B, co-injection of rRNA stimulated tRNA export and abolished the spike. We interpret this to mean that the stimulation of tRNA export by increasing concentrations of tRNA was precluded by the saturation of common retention sites with co-injected rRNA. This result indicates that both tRNA and rRNA stimulate tRNA export by titrating a common set of retention sites.

DISCUSSION

The kinetic results described provide insight into two intermediates along the tRNA export pathway. First, the saturable intermediate in the ribosome export pathway is not limiting for tRNA export. This suggests that tRNA and ribosomal subunit export pathways contain at least one distinct step. Second, the kinetics indicate that the rate-limiting step in tRNA export is a transient association with intranuclear retention sites. The titration of these sites increases instead of competes the rate of tRNA export. The first finding extends the work of Jarmolowski et al.(14) , who reported that RNA type-specific factors are involved in the export of tRNA, mRNA, and snRNAs. It may turn out that the export of each type of RNA, including tRNA, rRNA, U snRNA, signal recognition particle RNA, and mRNA, is mediated by at least one specific transport factor, although common intermediates, such as those associated with the nuclear pore complex will probably be shared. Ordering and characterizing each intermediate along the targeting and translocation pathways of different RNAs is an important challenge. It is relevant to note that the import of certain U small nuclear ribonucleoprotein particles appears to be mediated by specific factor(s) that are distinct from those employed during the import of most nuclear proteins(9, 10) .

The idea that nuclear transport can be regulated by retention is not a new idea. NLS anchoring in the cytoplasm is an established mechanism for regulated protein import(1) . Also, intranuclear retention has been proposed to be a general mechanism for controlling the export of proteins from nuclei(34) . Beginning with the initiation of transcription and proceeding through their processing and maturation, nascent RNAs are associated with large intranuclear structures, including nucleoli and speckles (reviewed in (36) ). In the nucleus, the localization of RNAs in supramolecular assemblies precludes their free diffusion and functions to retain immature RNAs in the nucleus (but see (35) ). Examples of RNA-specific retention during normal cell growth and in viral infections are documented(29) . In the case of tRNA export, Haselbeck and Greer (27) recently showed that microinjected intron-containing tRNAs were retained in the oocyte nucleus, while spliced tRNAs were exported.

In this study, evidence for transient RNA retention came from experiments that suggest the existence of an intermediate that normally functions to limit the rate of tRNA export. According to this model, when subsaturating concentrations of tRNA are microinjected into nuclei, the concentration of free tRNA that is accessible to the export apparatus is determined by the K of tRNA for a limiting set of intranuclear retention sites. As the retention sites become filled with increasing concentrations of injected tRNA, a higher proportion of the injected tRNA will be unbound and free to be exported, hence the stimulation. Because the range of tRNA concentrations over which stimulation of transport occurs is less than half that needed to significantly compete tRNA export (Fig. 5), we conclude that the affinity of tRNA for the retention sites is higher than for the saturable factor.

Because saturating concentrations of 40 S subunits and rRNA stimulate rather than compete tRNA export, we conclude that tRNA export does not employ the saturable factor(s) that is required for ribosome export. In the converse experiment, in which 40 S subunit export was challenged with saturating concentrations of tRNA, a different result was obtained. Here, high concentrations of tRNA partially compete the rate of 40 S subunit export. Previously, Dargemont and Kühn (13) reported that tRNA competed the export of mRNA, and subsequently, Jarmolowski et al.(14) showed that tRNA inhibited the rate of mRNA export, but only at relatively high concentrations. Although more analysis is needed, the simplest explanation for these data is that nonphysiologically high concentrations of microinjected tRNA bind in a pseudospecific (low affinity) fashion with factors that normally bind with physiologically high affinities to mRNA and 40 S subunits. Pseudospecific competitive inhibition would also explain the observed inhibition of tRNA and mRNA export by high concentrations of homopolymeric RNAs(14) . We cannot, however, rule out the interpretation that the effect of high concentrations of tRNA on mRNA and rRNA export indicates a common transport intermediate.

The putative intranuclear retention sites can be titrated with a range of different RNA substrates. Thus, tRNA, 40 S ribosomes, and total rRNA were each able to stimulate the percent rate of tRNA export. We also found that at 20 min post-injection, 5`-mGpppG-capped mRNA (8 µM) stimulated tRNA export to a statistically significant extent (125 ± 0.8% of control tRNA export in four experiments). Bovine serum albumin (20 µM) and double-stranded pBR322 DNA (1 µM) had no statistically significant effect. In the future, the tRNA export stimulation assay will provide a quantitative means to assess the specificity of the sites for different RNAs.

What are these intranuclear RNA retention sites? At one extreme, we cannot rule out that microinjected RNAs bind artifactually to sites that are normally inaccessible to endogenously transcribed and processed RNAs. However, the fact that the putative retention sites titrate at lower tRNA concentrations than those required to compete tRNA export argues that they are specific. The sites might represent component(s) of the normal transcriptional and post-transcriptional retention mechanisms. A more provocative explanation draws on a potential parallel between protein import and RNA export. During protein import, the binding of NLS-containing proteins to cytoplasmic NLS receptors is believed to be the saturable step(1, 2) . The rate-limiting step for protein import occurs after the substrate is targeted to the pore complex and is commonly assumed to involve some intermediate during the translocation process(37) . NLS-containing proteins, adsorbed to colloidal gold particles, have been observed to cluster about nuclear pore complex-associated filaments that extend into the cytoplasm(37, 38) . It is reasonable to assume that these perinuclear docking sites are general karyophile-binding sites and represent a later step along the import pathway of most nuclear proteins.

The results presented here provide kinetic evidence that tRNA export, like protein import, has different saturable and rate-limiting steps. The saturable step is tRNA-specific, whereas the rate-limiting step is common to tRNA, rRNA, and mRNA. The rate-limiting step, which we postulate involves dissociation from retention sites, might represent pore complex-associated RNA-binding sites. Unfortunately, these kinetic experiments provide no insight as to the intranuclear location of the RNA retention sites or as to whether they occur prior to or after pore complex targeting. In analogy to the putative cytoplasmic docking filaments, Fabre et al.(31) recently presented evidence for the presence of general RNA-binding proteins at the nuclear pore complex. Specifically, the yeast nucleoporin Nup145p is a member of a family of pore complex proteins, is required for mRNA export, and binds specifically to poly(G) RNA. The authors propose that RNAs transiently associate with these RNA-binding proteins during export. We think it is an attractive hypothesis that access of substrates to the pore complex is restricted at both faces by substrate docking sites that serve to regulate access to the translocation apparatus.

This model predicts that mature tRNAs are released from their processing components and become associated with the nuclear envelope. Here, they transiently bind general RNA docking sites. Release from these sites to the translocation channel is rate-limiting for tRNA. Titration of the retention sites with microinjected tRNA, mRNA, or rRNA allows unbound tRNAs to bypass the rate-limiting step and directly access the translocation channel. Under these conditions, export is still limited by the availability of saturable export factors that are specific for tRNA export.

There is no kinetic evidence for titratable retention sites that limit the rate of protein import or, for that matter, any transport substrate other than tRNA. This does not rule out the existence of such sites because kinetic evidence is only obtainable under certain conditions. Specifically, stimulation of transport by the titration of retention sites would be apparent only if the retention sites became titrated at substrate concentrations that are lower than those needed to saturate the substrate-specific export factors. If the substrate competes transport at substrate concentrations that are lower than those that would titrate the retention sites, then any stimulatory effects would be masked by prior competition. In this regard, tRNA export may be unique in that the process becomes saturated at concentrations in excess of those needed to show stimulation. Thus, while retention sites may be a general phenomenon in nuclear import and export, their existence may not always be revealed using the transport stimulation criterion.

FOOTNOTES

*

This work was supported by a United States Public Health Service FIRST award (to D. S. G.) and a National Institutes of Health postdoctoral fellowship (to N. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Biology, Vassar College, Poughkeepsie, NY 12601.

¶

To whom correspondence should be addressed. Tel.: 716-275-3890; Fax: 716-275-2070.

()

The abbreviations used are: NLSs, nuclear localization signals; snRNA, small nuclear RNA.

ACKNOWLEDGEMENTS

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