Elisa Izaurralde

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Rans nucleotide‐bound state is determined by the chromatin‐bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin‐β has previously been shown to release importin‐α from ‐β during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin‐α and ‐β, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.

TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.

The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin‐β mediates translocation via direct interaction with NPC components and carries importin‐α with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin‐β which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin‐β for its multiple partners. Ran and importin‐α respectively require residues 1–364 and 331–876 of importin‐β for binding. Thus, RanGTP‐mediated release of importin‐α from importin‐β is likely to be an active displacement rather than due to simple competition between Ran and importin‐α for a common binding site. Importin‐β has at least two non‐overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin‐β from the NPC. Importin‐β fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin‐β mutants are very efficient inhibitors of NLS‐dependent protein import. Surprisingly, however, they also inhibit M9 signal‐mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES‐containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.

Identification of a tRNA-Specific Nuclear Export Receptor

In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.

Dbp5, a DEAD‐box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p

Dbp5 is a DEAD‐box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope‐bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N‐terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu→Gln change in the conserved DEAD‐box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA‐dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus

The constitutive transport element (CTE) of the simian type D retroviruses overcomes nuclear retention and allows nuclear export of unspliced viral RNAs by recruiting TAP, a host factor which is thought to be required for export of cellular mRNAs. In this report, we show that the first 372 amino acid residues of TAP, comprising a stretch of leucine‐rich repeats, are both necessary and sufficient for binding to the CTE RNA and promoting its export to the cytoplasm. Moreover, like the full‐length protein, this domain migrates to the cytoplasm upon nuclear co‐injection with the CTE RNA. Together, these results indicate that the CTE‐binding domain includes the signals for nuclear export. We also describe a derivative of TAP that bears a triple amino acid substitution within the CTE‐binding domain and substantially reduces the export of mRNAs from the nucleus. This provides further evidence for a role for TAP in this process. Thus, the CTE‐binding domain of TAP defines a novel RNA‐binding motif which has dual functions, both recognizing the CTE RNA and interacting with other components of the nuclear transport machinery.

The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export

BACKGROUNDnA hallmark of retroviral gene expression is that unspliced retroviral genomic RNA is exported to the cytoplasm, whereas endogenous intron-containing cellular RNAs are usually retained in the nucleus. In complex retroviruses, such as human immunodeficiency virus-1 (HIV-1), nuclear export is accomplished by the interaction of a virally encoded protein, Rev, with a cis-acting RNA element, the Rev-responsive element (RRE). In type D retroviruses, such as the simian retrovirus type 1 (SRV-1), however, genomic RNA is exported by cellular factor(s) that interact with a conserved cis-acting RNA element, the constitutive transport element (CTE).nnnRESULTSnWe found that the CTE was exported in a specific and saturable fashion from Xenopus oocyte nuclei. When inserted into the intron of an adenovirus-derived pre-mRNA, the CTE did not affect splicing efficiency but promoted the nuclear export of the excised intron lariat that is normally retained within the nucleus. Export of CTE-containing RNAs to the cytoplasm was not affected by the heterogeneous nuclear ribonucleoprotein A1 or an excess of peptides corresponding to the Rev nuclear export signal. Microinjection of saturating amounts of CTE RNA did not affect tRNA export or Rev-mediated export but did inhibit mRNA export. CTE-mediated export was found to be dependent on Ran-mediated GTP hydrolysis.nnnCONCLUSIONnThe Rev-RRE system and the CTE direct intron-containing RNAs to distinct export pathways. Although previous data have suggested that Rev uses the same export pathway as uracil-rich small nuclear RNAs and 5S ribosomal RNA, the CTE seems to interact with evolutionarily conserved factors that are essential for cellular mRNA export.

Highly preferential nucleation of histone H1 assembly on Scaffold-associated regions

Scaffold-associated regions (SARs) are A + T-rich DNA regions of several hundred base-pairs that are known to bind specifically to nuclear or metaphase scaffolds. Surprisingly, histone H1 specifically associates with SARs. Under conditions of high co-operativity, at input ratios of H1 to DNA up to 15% (w/w), histone H1 binds preferentially to those DNA molecules harboring a SAR, leaving the non-SAR fragments free. Our experiments identify SARs as cis-acting sequences that nucleate co-operative H1 assembly along the SAR into the flanking non-SAR DNA. Experiments with simple DNA polymers implicate homopolymeric oligo(dA).oligo(dT) tracts in preferential histone H1 assembly. The homopolymer oligo(dA).oligo(dT) is, above a critical length of 130 base-pairs, a highly specific nucleator of H1 assembly. SARs may control the conformation of chromatin domains via a regulated H1 assembly and set up the potential transcriptional repertoire of the cell.

Interaction of DNA with nuclear scaffolds in vitro

We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds.

Coordination of tRNA nuclear export with processing of tRNA

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5 and 3 ends, posttranscriptional addition of the 3 CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5 and 3 ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5 and 3 terminal ends, and the TpsiC loop are critical for exportin-t binding.