mRNA Export Machinery Research Articles

Nuclear export assays for poly(A) RNAs

Nuclear export of mRNAs is a central step in eukaryotic gene expression. A defect in bulk poly(A) RNA export can be caused either by a direct disruption of the mRNA export machinery or by an indirect effect on mRNA biogenesis. One example of interference with the mRNA export pathway is viral–host interactions involving mRNA export factors. VSV M protein binds the mRNA export factor Rae1 that is in complex with Nup98, resulting in nuclear retention of mRNAs. To study regulation of mRNA export, we review here two useful methodologies, which include a reporter gene assay and oligo(dT) in situ hybridization. In a reporter gene assay one can assess up-regulation or down-regulation of gene expression that can occur at different levels, including transcription, mRNA processing, mRNA export, and translation. An effect on mRNA export can then be identified by determining the intracellular distribution of poly(A) RNA using oligo(dT) in situ hybridization. Reporter gene assays are quick, relatively simple and can thus be used in primary highthroughput screenings. To further pinpoint disruption of mRNA export, oligo(dT) in situ hybridization can be used. Since it is a more laborious methodology it is more suitable for a secondary screening. We also review here a combination of oligo(dT) in situ hybridization with immunofluorescence for simultaneous localization of endogenous or ectopically expressed proteins. Altogether, these assays are valuable tools for identifying major regulatory effects on mRNA nuclear export.

The UL69 Transactivator Protein of Human Cytomegalovirus Interacts with DEXD/H-Box RNA Helicase UAP56 To Promote Cytoplasmic Accumulation of Unspliced RNA

The UL69 gene product of human cytomegalovirus belongs to a family of regulatory proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.

Hsp16p Is Required for Thermotolerance in Nuclear mRNA Export in Fission Yeast Schizosaccharomyces pombe

Export of mRNA from the nucleus to the cytoplasm is one of the essential steps for eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, heat shock stress at 42 degrees C causes block of mRNA export from the nucleus. We now report that saline and ethanol stresses also inhibit nuclear mRNA export, resulting in accumulation of bulk poly (A)+ RNA, as well as a specific mRNA, in the nucleus. Under stressed conditions, an mRNA export receptor Mex67p relocates to the nucleolus from the nuclear periphery and this relocation is closely correlated with inhibition of mRNA export by the stresses. Pretreatment of cells with a mild saline stress induced thermotolerance in mRNA export in a similar manner seen with mild heat pretreatment and protected mRNA export machinery from the subsequent severe heat shock. In contrast, mild ethanol stress could not induce the thermotolerance in mRNA export, suggesting that the stress response induced by ethanol differs from that induced by saline and heat shock stresses. In addition, we found that the Spc1p MAPK pathway is involved in induction of thermotolerance in mRNA export. Of the downstream targets for Spc1p, the Atf1p transcription factor was essential for induction of thermotolerance in mRNA export. We also found that Hsp16p, the expression of which is controlled by Atf1p, is involved in acquisition of thermotolerance in mRNA export in S. pombe.

Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery.

Centrins are calmodulin-like proteins that function in the duplication of microtubule-organizing centres. Here we describe a new function of the yeast centrin Cdc31. We show that overproduction of a sequence, termed CID, in the carboxy-terminal domain of the nuclear export factor Sac3 titrates Cdc31, causing a dominant-lethal phenotype and a block in spindle pole body (SPB) duplication. Under normal conditions, the CID motif recruits Cdc31 and Sus1 (a subunit of the SAGA transcription complex) to the Sac3-Thp1 complex, which functions in mRNA export together with specific nucleoporins at the nuclear basket. A previously reported cdc31 temperature-sensitive allele, which is neither defective in SPB duplication nor Kic1 kinase activation, induces mRNA export defects. Thus, Cdc31 has an unexpected link to the mRNA export machinery.

Differential Export Requirements for Shuttling Serine/Arginine-type mRNA-binding Proteins

Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins. Here, we present the characterization of Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae. The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3. Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10. Hrb1 binds to poly(A)+ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs. Interestingly, by comparing the export requirements of all three SR proteins we find similarities but also striking differences. While the export of all three proteins is dependent on the export of mRNAs in general, as no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), we find specific requirements for components of the THO complex, involved in transcription elongation. While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins. These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1.

Sus1, a Functional Component of the SAGA Histone Acetylase Complex and the Nuclear Pore-Associated mRNA Export Machinery

Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery.

The Mtr2-Mex67 NTF2-like Domain Complex: STRUCTURAL INSIGHTS INTO A DUAL ROLE OF Mtr2 FOR YEAST NUCLEAR EXPORT

The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.

UAP56 levels affect viability and mRNA export in Caenorhabditis elegans.

Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi's resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A', CstF50).

Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery.

Nuclear export of mRNA is mediated by a complex machinery of RNA-binding proteins that recognizes and routes mRNAs through a messenger ribonucleoprotein (mRNP) network. The full spectrum of mRNA cargoes for any dedicated mRNA export factor is unknown. We identified the mRNAs that bind two conserved yeast mRNA export factors, Yra1 (refs. 1-5) and Mex67 (refs. 6,7), on a genome-wide scale and determined their level of binding. Yra1 and Mex67 bind approximately 1,000 and 1,150 mRNAs, respectively, corresponding to almost 20% of the yeast genome and roughly 36% of all transcriptional events each. The binding level of Yra1 targets is related to their transcriptional frequency, but that of Mex67 targets is not. Yra1-bound transcripts are enriched in mRNAs that are regulated by a number of transcription factors. Yra1- and Mex67-bound populations also show enrichment of mRNAs encoding distinct functional classes of proteins, some of which are regulated by these transcription factors. We determined that one such transcription factor, Abf1 (refs. 8-10), associates with Yra1. These results indicate a previously unidentified specificity of mRNA export factors, which coordinates the export of transcriptionally co-regulated, functional classes of transcripts, perhaps through interactions with the transcriptional machinery.

The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores.

Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs. Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p. In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation. In addition, Sac3p binds to the mRNA exporter Mex67p-Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC). Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects. Taken together, our data suggest that the novel Sac3p-Thp1p complex functions by docking the mRNP to specific nucleoporins at the nuclear entrance of the NPC.

The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans.

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.