Ratcheting mRNA out of the nucleus

Export of mature mRNA to the cytoplasm is the culmination of the nuclear portion of eukaryotic gene expression. After transport-competent mature mRNP export complexes are formed in the nucleus, their passage through nuclear pore complexes (NPCs) is facilitated by the Mex67:Mtr2 heterodimer. At the NPC cytoplasmic face, mRNP remodeling prevents its return to the nucleus and so functions as a molecular ratchet imposing directionality on transport. In budding yeast, recent work suggests that the DEAD-box helicase Dbp5 remodels mRNPs at the NPC cytoplasmic face by removing Mex67 and that the Dbp5 ATPase is activated by Gle1 and inositol hexaphosphate (IP(6)).     

The nuclear basket mediates perinuclear mRNA scanning in budding yeast

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.     

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.     

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.     

The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance.     

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153.     

Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.     

Crystal Structure of the N-Terminal Domain of Nup358/RanBP2

Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95 Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPRs), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC.     

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.     

Mechanisms of nuclear mRNA export: A structural perspective

Export of mRNA from the nucleus to the cytoplasm is a critical process for all eukaryotic gene expression. As mRNA is synthesized, it is packaged with a myriad of RNA‐binding proteins to form ribonucleoprotein particles (mRNPs). For each step in the processes of maturation and export, mRNPs must have the correct complement of proteins. Much of the mRNA export pathway revolves around the heterodimeric export receptor yeast Mex67?Mtr2/human NXF1?NXT1, which is recruited to signal the completion of nuclear mRNP assembly, mediates mRNP targeting/translocation through the nuclear pore complex (NPC), and is displaced at the cytoplasmic side of the NPC to release the mRNP into the cytoplasm. Directionality of the transport is governed by at least two DEAD‐box ATPases, yeast Sub2/human UAP56 in the nucleus and yeast Dbp5/human DDX19 at the cytoplasmic side of the NPC, which respectively mediate the association and dissociation of Mex67?Mtr2/NXF1?NXT1 onto the mRNP. Here we review recent progress from structural studies of key constituents in different steps of nuclear mRNA export. These findings have laid the foundation for further studies to obtain a comprehensive mechanistic view of the mRNA export pathway.     

The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events

Messenger RNA (mRNA) export involves the unidirectional passage of ribonucleoprotein particles (RNPs) through nuclear pore complexes (NPCs), presumably driven by the ATP-dependent activity of the DEAD-box protein Dbp5. Here we report that Dbp5 functions as an RNP remodeling protein to displace the RNA-binding protein Nab2 from RNA. Strikingly, the ADP-bound form of Dbp5 and not ATP hydrolysis is required for RNP remodeling. In vivo studies with nab2 and dbp5 mutants show that a Nab2-bound mRNP is a physiological Dbp5 target. We propose that Dbp5 functions as a nucleotide-dependent switch to control mRNA export efficiency and release the mRNP from the NPC.     

Hot spot formation in the nuclear envelope of oocytes in response to steroids.

A Glucocorticoid-sensitive cell rapidly responds to hormone stimulation with bidirectional exchange of specific macromolecules between cytosol and nucleus. Glucocorticoid-initiated macromolecules (GIMs) must overcome the nuclear envelope (NE) to enter or leave the nucleus. GIM translocation occurs through nuclear pore complexes (NPCs) that span the NE. We investigated the question whether transport of GIMs through NPCs occurs random or involves selected groups of NPCs (hot spots). Glucocorticoid receptors were expressed in Xenopus laevis oocytes and GIM transport was activated by triamcinolone acetonide, a potent synthetic glucocorticoid analogon. Glucocorticoid receptors associated with the NE and the chromatin were identified using western blot analysis and, at single molecule level, atomic force microscopy. Fluorescence-labeled dextran was used to describe passive NE permeability. We observed that after hormone injection (i) small GIMs, most likely GRs, localize within seconds on both sides of the NE. (ii) large GIMs, most likely ribonucleoproteins, localize within minutes on NPCs at the nucleoplasmic side (iii) both small and large GIMs accumulate on selected NPC clusters (iv) NE permeability transiently decreases when GIMs attach to NPCs. We conclude that GIM transport across the nuclear barrier does not randomly take place but is carried out by a selected population of NPCs.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.