Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

Nuclear export of mRNA.

Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems. Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions. Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm.     

Nuclear PRP20 protein is required for mRNA export.

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.     

Nuclear export and cytoplasmic maturation of ribosomal subunits

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.     

REF-ereeing the cytoplasmic fate of mRNA via nuclear export

The C. elegans sex-determining gene tra-2 is subject to multiple forms of regulation. A report in the June 4 issue of Molecular Cell now shows that proteins associated with the tra-2 mRNA determine its pathway of nuclear export and influence its cytoplasmic fate. These findings demonstrate an additional level of control and link nuclear export to the regulation of sexual development.     

Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex

In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.     

A Sm-like protein complex that participates in mRNA degradation

In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming.     

Engagement of tumor necrosis factor mRNA by an endotoxin-inducible cytoplasmic protein.

BACKGROUND: Tumor necrosis factor (TNF) production by macrophages plays an important role in the host response to infection. TNF-alpha gene expression in RAW 264.7 macrophages is predominantly regulated at the translational level. A key element in this regulation is an AU-rich (AUR) sequence located in the 3' untranslated region (UTR) of TNF mRNA. In unstimulated macrophages, the translation of TNF mRNA is inhibited via this AUR sequence. Upon stimulation with LPS, this repression is overcome and translation occurs. In this study, we attempted to identify cellular proteins that interact with the AUR sequence and thereby regulate TNF mRNA translation. MATERIALS AND METHODS: RNA probes corresponding to portions of TNF mRNA 3' UTR were synthesized. These labeled RNAs were incubated with cytoplasmic extracts of either unstimulated or lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The RNA/protein complexes formed were analyzed by gel retardation. Ultraviolet (UV) cross-linking experiments were performed to determine the molecular weight of the proteins involved in the complexes. RESULTS: TNF mRNA AUR sequence formed two complexes (1 and 2) of distinct electrophoretic mobilities. While the formation of complex 1 was independent of the activation state of the macrophages from which the extracts were obtained, complex 2 was detected only using cytoplasmic extracts from LPS-stimulated macrophages. Upon UV cross-linking, two proteins, of 50 and 80 kD, respectively, were capable of binding the UAR sequence. The 50-kD protein is likely to be part of the LPS-inducible complex 2, since its binding ability was enhanced upon LPS stimulation. Interestingly, complex 2 formation was also triggered by Sendaï virus infection, another potent activator of TNF mRNA translation in RAW 264.7 macrophages. In contrast, complex 2 was not detected with cytoplasmic extracts obtained from B and T cell lines which are unable to produce TNF in response to LPS. Protein tyrosine phosphorylation is required for LPS-induced TNF mRNA translation. Remarkably, the protein tyrosine phosphorylation inhibitor herbimycin A abolished LPS-induced complex 2 formation. Complex 2 was already detectable after 0.5 hr of LPS treatment and was triggered by a minimal LPS dose of 10 pg/ml. CONCLUSIONS: The tight correlation between TNF production and the formation of an LPS-inducible cytoplasmic complex suggests that this complex plays a role in the translational regulation of TNF mRNA.     

TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.     

Characterization of the betaherpesviral pUL69 protein family reveals binding of the cellular mRNA export factor UAP56 as a prerequisite for stimulation of nuclear mRNA export and for efficient viral replication

UL69 of human cytomegalovirus (HCMV) encodes a pleiotropic transactivator protein and has a counterpart in every member of the Herpesviridae family thus far sequenced. However, little is known about the conservation of the functions of the nuclear phosphoprotein pUL69 in the homologous proteins of other betaherpesviruses. Therefore, eukaryotic expression vectors were constructed for pC69 of chimpanzee cytomegalovirus, pRh69 of rhesus cytomegalovirus, pM69 of murine cytomegalovirus, pU42 of human herpesvirus 6, and pU42 of elephant endotheliotropic herpesvirus. Indirect immunofluorescence experiments showed that all pUL69 homologs expressed by these vectors were localized to the cell nucleus. Coimmunoprecipitation experiments identified homodimerization as a conserved feature of all homologs, whereas heterodimerization with pUL69 was restricted to its closer relatives. Further analyses demonstrated that pC69 and pRh69 were the only two homologs that functioned, like pUL69, as viral-mRNA export factors. As we had reported recently that nucleocytoplasmic shuttling and interaction with the cellular DExD/H-box helicases UAP56 and URH49 were prerequisites for the nuclear-mRNA export activity of pUL69, the homologs were characterized with regard to these properties. Heterokaryon assays demonstrated nucleocytoplasmic shuttling for all homologs, and coimmunoprecipitation and mRNA export assays revealed that the interaction of UAP56 and/or URH49 with pC69 or pRh69 was required for mRNA export activity. Moreover, characterization of HCMV recombinants harboring mutations within the N-terminal sequence of pUL69 revealed a strong replication defect of viruses expressing pUL69 variants that were deficient in UAP56 binding. In summary, homodimerization and nucleocytoplasmic shuttling activity were identified as conserved features of betaherpesviral pUL69 homologs. UAP56 binding was shown to represent a unique characteristic of members of the genus Cytomegalovirus that is required for efficient replication of HCMV.     

Nuclear export of simian immunodeficiency virus Vpx protein

Lentiviruses, human immunodeficiency viruses (HIVs), and simian immunodeficiency viruses (SIVs) are distinguished from oncoretroviruses by their ability to infect nondividing cells such as macrophages. Retroviruses must gain access to the host cell nucleus for replication and propagation. HIV and SIV preintegration complexes (PIC) enter nuclei after traversing the central aqueous channel of the limiting nuclear pore complex without membrane breakdown. Among the nucleophilic proteins, namely, matrix, integrase, Vpx, and Vpr, present in HIV type 2/SIV PIC, Vpx is implicated in nuclear targeting and is also available for incorporation into budding virions at the plasma membrane. The mechanisms of these two opposite functions are not known. We demonstrate that Vpx is a nucleocytoplasmic shuttling protein and contains two novel noncanonical nuclear import signals and a leptomycin B-sensitive nuclear export signal. In addition, Vpx interacts with the cellular tyrosine kinase Fyn through its C-terminal proline-rich motif. Furthermore, our data indicate that Fyn kinase phosphorylates Vpx and regulates its export from nucleus. Replacement of conserved tryptophan residues within domain 41 to 63 and tyrosine residues at positions 66, 69, and 71 in Vpx impairs its nuclear export, virion incorporation, and SIV replication in macrophages. Nuclear export is essential to ensure the availability of Vpx in the cytoplasm for incorporation into virions, leading to efficient viral replication within nondividing cells.