Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA 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.     

REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.     

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.     

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.     

Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.     

Crp79p,like Mex67p,is an auxiliary mRNA export factor in Schizosaccharomyces pombe

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.     

Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.     

The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p

Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export. Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271). Using RNA interference experiments in C. elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes. No embryonic or progeny abnormality was observed in functional knockout of C15H11.6. Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1. Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim. The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins. This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex. In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex. Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.     

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 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.