Nucleocytoplasmic transport: cargo trafficking across the border

Transport of macromolecules between the cytoplasm and the nucleus is mediated by at least three different classes of soluble transport receptors, members of the importin-beta protein family, the Mex67/Tap family and the small nuclear transport factor 2 (NFT2). All nuclear transport factors can bidirectionally traverse the nuclear pore complex through specific interactions with phenylalanine/glycine-rich nuclear pore complex components. Recent kinetic and structural analyses revealed novel insight into the details of these interactions. In addition, new biochemical and genetic studies have dramatically improved our understanding of ribosomal and messenger RNA export, unveiling a tight coupling between RNA processing and transport.     

A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.     

A new view of mRNA export: separating the wheat from the chaff

Current models for the export of messenger RNA share the notion that the highly abundant class of nuclear RNA-binding proteins--the hnRNP proteins--have a key role in exporting RNA. But recent studies have led to a new understanding of several non-hnRNP proteins, including SR proteins and the conserved mRNA export factor ALY, which are recruited to the mRNA during pre-mRNA splicing. These studies, together with older work on hnRNP particles and assembly of the spliceosome, lead us to a new view of mRNA export. In our model, the non-hnRNP factors form a splicing-dependent mRNP complex that specifically targets mature mRNA for export, while hnRNP proteins retain introns in the nucleus. A machinery that is conserved between yeast and higher eukaryotes functions to export the mRNA.     

The mRNA export pathway in plants

A double lipid bilayer separating the nucleus from the cytoplasm, termed the nuclear envelope, is a defining feature of eukaryotes. Nucleocytoplasmic transport of macromolecules through the nuclear pores enables fine-tuned regulation of biologic processes. All mature mRNAs are delivered to the cytoplasm from the nucleus via an mRNA export pathway. Much work has been done in yeast and animals to study the machinery of mRNA export. However, until recently, research on plant mRNA export has been quite limited. Genetic, bioinformatic, and biochemical investigations have expanded our understanding of the mRNA export process in plants. Here, we review recent progress that has been made elucidating the components of the mRNA export pathway in plants. MOS3 (MODIFIER OF SNC1, 3) /AtNup96 and AtNup160 are both components of the highly conserved Nup107–160 nucleoporin complex and were shown to play key roles in mRNA export. MOS11 (MODIFIER OF SNC1, 11), which is homologous to the RNA helicase enhancer CIP29 in human, was recently found to be involved in the same pathway as MOS3. A DEAD Box RNA helicase, LOS4 (low expression of osmotically responsive genes 4) was also found to play a role in the mRNA export process, putatively by carrying mRNA molecules through the nuclear envelope. Recently, a protein complex homologous to the yeast TREX-2 complex was also found to play important roles in mRNA export in plants. It appears that most players in the mRNA export pathway are highly conserved among plants, yeast and animals.     

The nuclear transport machinery in Caenorhabditis elegans: A central role in morphogenesis

Our full understanding of the various roles for the nuclear transport machinery has come from a variety of model organisms including yeast, nematodes, fruit flies and vertebrates. Using the nematode Caenorhabditis elegans, it has been shown that the karyopherin family of nuclear transporters and the components of the Ran cycle have roles not only in nuclear protein transport, but also in mitotic spindle formation and regulation, and in nuclear envelope assembly. These studies have also demonstrated a role for nuclear transport factors in cellular differentiation and development, particularly for the formation of germ cells. This review highlights the small number of studies in C. elegans that have been critical to our understanding of this important cellular process.     

The application of yeast hybrid systems in protein interaction analysis

Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein–protein, protein–DNA and protein–RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein–protein interactions, cytosol protein–protein interactions and membrane protein–protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

A panoramic view of yeast noncoding RNA processing

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.     

Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport

To extend our understanding of the mechanism by which the nuclear pore complex (NPC) mediates macromolecular transport across the nuclear envelope we have focused on defining the composition and molecular organization of the yeast NPC. Peptide sequence analysis of a polypeptide with a M(r) of approximately 100,000 present in a highly enriched yeast NPC fraction identified a novel yeast nucleoporin we term Nup120p. Nup120p corresponds to the open reading frame (ORF) YKL057c identified by the yeast genome sequencing project. The ORF predicts a protein with a calculated molecular mass of 120.5 kD containing two leucine zipper motifs, a short coiled-coil region and limited primary sequence similarity to Nup133p. Nup120p was localized to the NPC using a protein A-tagged chimera in situ by indirect immunofluorescence microscopy. Deletion of the NUP120 gene caused clustering of NPCs at one side of the nuclear envelope, moderate nucleolar fragmentation and slower cell growth. Transfer of nup120 delta cells to 37 degrees C resulted in the nuclear accumulation of poly(A)+ mRNA, extensive fragmentation of the nucleolus, spindle defects, and cell death.     

Nuclear RNA export in yeast.

Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.     

The search for the primary function of the Ran GTPase continues

It has been nearly 20 years since the discovery of the first component of the Ran-GTPase pathway. Since then, nearly 100 articles, more than half of which have been published in the past three years, have reported the identification of additional components of the system and the existence of their structural and functional homologues in organisms ranging from yeast to man. The Ran system affects a vast array of nuclear processes including RNA metabolism, DNA replication, chromosome condensation and decondensation, and nucleocytoplasmic transport of protein and RNA. The current challenge is to identify the molecular targets that link the Ran-GTPase system to this collection o f nuclear processes.     

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.     

Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex

The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes.