Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes in the yellow mealworm Tenebrio molitor

The nucleus of vitellogenic oocytes of the yellow mealworm Tenebrio molitor contains a karyosphere that consists of condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters (IGCs)) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged 2??-O-Me(U)₂₂ methyl oligoribonucleotide probes, complementary to poly(A) tails of RNAs, revealed poly(A)⁺ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein A1 localized to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and some nucleoplasmic IGCs also contain the adapter protein Aly known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was colocalized with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner, whereas it was RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data provide evidence for the implication of nucleoplasmic IGCs in mRNA biogenesis and retention on the path to nuclear export.     

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.     

U2AF participates in the binding of TAP (NXF1) to mRNA.

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.     

SR splicing factors serve as adapter proteins for TAP-dependent mRNA export

The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export.     

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.     

Identification and characterization of the mouse nuclear export factor (Nxf) family members

TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.     

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.     

Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export

Human TAP and its yeast orthologue Mex67p are members of the multigene family of NXF proteins. A conserved feature of NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain). The NTF2-like domain of metazoan NXFs heterodimerizes with a protein known as p15 or NXT. A C-terminal region related to ubiquitin-associated domains (the UBA-like domain) is present in most, but not all NXF proteins. Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus. Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been established. Here we show that overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates the export of mRNAs that are otherwise exported inefficiently. This stimulation of mRNA export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain. Similar results are obtained when TAP/p15 heterodimers are directly tethered to the RNA export cargo. Our data indicate that formation of TAP/p15 heterodimers is required for TAP-mediated export of mRNA and show that the LRR domain of TAP plays an essential role in this process.     

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.     

NXF5, a novel member of the nuclear RNA export factor family, is lost in a male patient with a syndromic form of mental retardation

BACKGROUND: Although X-linked mental retardation (XLMR) affects 2%-3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development. RESULTS: We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain. CONCLUSIONS: A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.