Iain W. Mattaj

CRM1 Is an Export Receptor for Leucine-Rich Nuclear Export Signals

CRM1 is distantly related to receptors that mediate nuclear protein import and was previously shown to interact with the nuclear pore complex. Overexpression of CRM1 in Xenopus oocytes stimulates Rev and U snRNA export from the nucleus. Conversely, leptomycin B, a cytotoxin that is shown to bind to CRM1 protein, specifically inhibits the nuclear export of Rev and U snRNAs. In vitro, CRM1 forms a leptomycin B-sensitive complex involving cooperative binding of both RanGTP and the nuclear export signal (NES) from either the Rev or PKI proteins. We conclude that CRM1 is an export receptor for leucine-rich nuclear export signals and discuss a model for the role of RanGTP in CRM1 function and in nuclear export in general.

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.

Ran Induces Spindle Assembly by Reversing the Inhibitory Effect of Importin α on TPX2 Activity

Abstract The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin α, and is displaced from importin α by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin α. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.

A nuclear cap binding protein complex involved in pre-mRNA splicing

A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.

Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells),. The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose thatRCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.

Nucleocytoplasmic Transport: The Last 200 Nanometers

We wish to thank Susanne Bailer, Ed Hurt, Dirk Gorlich, and the members of our laboratory for critical comments on the manuscript. We thank Valerie Doye, Susanne Bailer, Roger Wepf, and Ed Hurt for Figure 4Figure 4 and Toby Gibson and Petra Riedinger for help with Figure 2Figure 2 and Figure 3Figure 3.

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.

Monomethylated cap structures facilitate RNA export from the nucleus

RNA export from the nucleus has been analyzed in Xenopus oocytes. U1 snRNAs made by RNA polymerase II were exported into the cytoplasm, while U1 snRNAs synthesized by RNA polymerase III, and therefore with a different cap structure, remained in the nucleus. Export of the polymerase II-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs carrying monomethylguanosine cap structures were rapidly exported, while hypermethylated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable splicing complexes was also significantly delayed by incorporation of a hypermethylated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus.

Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly.

Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97–Ufd1–Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97–p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

PHAX, a Mediator of U snRNA Nuclear Export Whose Activity Is Regulated by Phosphorylation

In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.