▪ Abstract The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin β-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin β-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

Transport between the cell nucleus and the cytoplasm.

microRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes Primary miRNA transcripts are initially processed to approximately 70-nucleotide (nt) stem-loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield approximately 22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner

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Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs

From sites of transcription in the nucleus to the outreaches of the cytoplasm, messenger RNAs are associated with RNA-binding proteins. These proteins influence pre-mRNA processing as well as the transport, localization, translation and stability of mRNAs. Recent discoveries have shown that one group of these proteins marks exon exon junctions and has a role in mRNA export. These proteins communicate crucial information to the translation machinery for the surveillance of nonsense mutations and for mRNA localization and translation.

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Messenger-RNA-binding proteins and the messages they carry.

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

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The Yeast Nuclear Pore Complex: Composition, Architecture, and Transport Mechanism

The yeast cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis. This review discusses recent advances in understanding the various signal transduction pathways that allow cells to monitor the state of the cell wall and respond to environmental challenges to this structure. The cell wall integrity signaling pathway controlled by the small G-protein Rho1 is principally responsible for orchestrating changes to the cell wall periodically through the cell cycle and in response to various forms of cell wall stress. This signaling pathway acts through direct control of wall biosynthetic enzymes, transcriptional regulation of cell wall-related genes, and polarization of the actin cytoskeleton. However, additional signaling pathways interface both with the cell wall integrity signaling pathway and with the actin cytoskeleton to coordinate polarized secretion with cell wall expansion. These include Ca2+ signaling, phosphatidylinositide signaling at the plasma membrane, sphingoid base signaling through the Pkh1 and -2 protein kinases, Tor kinase signaling, and pathways controlled by the Rho3, Rho4, and Cdc42 G-proteins.

Cell Wall Integrity Signaling in Saccharomyces cerevisiae

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

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Dbp5p, a cytosolic RNA helicase, is required for poly(A) + RNA export

An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A) 1 RNA in the nucleus at 37C120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export. The mechanism of export of mRNA from the nucleus to the cytoplasm is remarkably accommodating in the sense that an enormous variety of primary and secondary RNA structures are compatible with export. The fact that neither the 59methyl cap structure nor the 39poly(A) tail appears altogether essential for export (16) suggests that there is a mechanism in the nucleus which allows for RNA recognition independent of the sequence. It is likely that during or immediately after their synthesis, transcripts are quickly packaged into ribonucleoprotein particles (RNP) which enable them to gain access to the periphery of the nucleoplasm and interact specifically with componentsofnuclearpores,throughwhichtheysubsequently translocate (14, 42). If this itinerary is relatively uniform for most mRNAs, it is reasonable to postulate that there are factors which serve as RNA chaperones and/or normalize RNA secondary structure. Single-stranded RNAs spontaneously acquire an extensive secondary structure, which is subject to proteins with annealing activity (p53 and heterogeneous nuclearRNP[hnRNP]A1[24,25])andmeltingactivity(helicases [34]). Studies of the yeastSaccharomyces cerevisiaehave identified several proteins which, when mutated, lead to nuclear accumulation of poly(A) 1 RNA without inhibiting pre-mRNA splicing.Apartfromnucleoporins(8),theseincludescomponents of a nucleocytoplasmic GTPase cycle [the Cnr1/2p (Gsp1/2p) GTPases, Mtr1p (Prp20p), and Rna1p (6, 35)], a cytoplasmic protein (Mtr7p [34a]), the nucleolar protein Mtr3p (19), the nuclearproteinsMtr2p(19)andRat1p(1),andthenuclearhnRNP

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

Heat shock causes major positive and negative changes in gene expression, drastically alters the appearance of the nucleolus and inhibits rRNA synthesis. We here show that it causes many yeast nucleolar proteins, including the fibrillarin homolog Nop1p, to relocate to the cytoplasm. Relocation depends on several proteins implicated in mRNA transport (Mtrps) and is reversible. Two observations indicate, surprisingly, that disassembly results from a reduction in Ssa protein (Hsp70) levels: (i) selective depletion of Ssa1p leads to disassembly of the nucleolus; (ii) preincubation at 37 degrees C protects the nucleolus against disassembly by heat shock, unless expression of Ssa proteins is specifically inhibited. We observed that heat shock or reduction of Ssa1p levels inhibits protein import into the nucleus and therefore we propose that inhibition of import leads to disassembly of the nucleolus. These observations provide a simple explanation of the effects of heat shock on the anatomy of the nucleolus and rRNA transcription. They also extend understanding of the path of nuclear export. Since a number of nucleoplasmic proteins also relocate upon heat shock, these observations can provide a general mechanism for regulation of gene expression. Relocation of the hnRNP-like protein Mtr13p (= Npl3p, Nop3p), explains the heat shock sensitivity of export of average poly(A)+ RNA. Strikingly, Hsp mRNA export appears not to be affected.

Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export.

An Unexpected Link between the Secretory Path and the Organization of the Nucleus

mRNA export involves association of mRNAs with nucleoplasmic proteins, delivery to the nuclear pore complex, translocation to the cytoplasm, and reimport of recycling components. Many yeast mutants inhibit mRNA export, but there is little information concerning the RNA carriers and steps of transport that they affect. The hnRNP/serine-arginine-rich-like protein, Npl3p/Mtr13p, binds poly(A)+ RNA and shuttles between the nucleus and cytoplasm. Its export accelerates on inhibition of RNA synthesis. In vivo tests show that its export requires two proteins with putative leucine-rich nuclear export signals: Gle1p, Mex67p, and several additional nuclear and nuclear pore complex-associated proteins. Surprisingly, a nonnuclear pool of an import factor (the importin α homologue, Srp1p) is also required. Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution. A crm1 mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export. Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo. Thus, Npl3p export requires only a subset of proteins implicated in mRNA export, suggesting that more than one mRNA export path exists. A distinct group of mutants, including a mutation of a member of the importin β superfamily, inhibits Npl3p reimport from the cytoplasm.

Yan Liu

Papers

Dbp5p, a cytosolic RNA helicase, is required for poly(A) + RNA export

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export.

An Unexpected Link between the Secretory Path and the Organization of the Nucleus

A Crm1p-independent nuclear export path for the mRNA-associated protein, Npl3p/Mtr13p.