Export Adaptor Protein Research Articles

Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein

IntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathway using NS2/NEP as an export adaptor protein. NEP is a 14 kDa protein and diffusely localizes in the nucleus and cytoplasm. Upon binding to the NLS motif of M1, NEP inhibits the nuclear accumulation of M1 and promotes the nuclear export of M1-viral RNP complex. However, the detail mechanism by which NEP binds to M1 only in the nucleus remains unclear.MethodsTo visualize the interaction of NEP with M1 in the formation of vRNP export complexes, we performed in situ proximity ligation assays. The close proximity of N-terminal and C-terminal domains of NEP was tested by split Renilla luciferase complementation assays in which the N-terminal and C-terminal fragments of Renilla luciferase were fused to the N-terminus and C-terminus of NEP, respectively.Results and discussionWe found that the intramolecular interaction of NEP inhibits the interaction of NEP with M1. The intramolecular interaction of NEP was mediated through the interaction of the N-terminal NES motif with the M1-binding domain at the C-terminus. By adding leptomycin B, a potent inhibitor of CRM1, the interaction of NEP with M1 was impaired. These results suggest that CRM1 disrupts the intramolecular interaction of NEP by recognizing the NES motif at the N-terminus of NEP, thereby promoting the interaction of NEP with M1. We also found that NEP mutant deficient in the intramolecular interaction was co-localized with M1 at the plasma membrane and did not show nuclear localization with M1. Based on these results, we propose that the intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1.

Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs.

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.

GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72-) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ(2) P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ(2) P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.

Highly Pathogenic Avian Influenza Virus Nucleoprotein Interacts with TREX Complex Adaptor Protein Aly/REF

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

Biochemical Analysis of the Complex between the Tetrameric Export Adapter Protein Rec of HERV-K/HML-2 and the Responsive RNA Element RcRE pck30

The RNA export adaptor protein Rec, encoded for by the human endogenous retrovirus HERV-K/HML-2 elements, binds to the Rec responsive element (RcRE) located in the 3' untranslated region of HERV-K/HML-2 transcripts. Binding allows the nucleocytoplasmic export of unspliced viral RNA, thereby overcoming host restriction. Chemical probing of the secondary structure of the RcRE corroborated the theory that the RcRE forms a complex folded structure with seven stem-loop regions. Laser-induced liquid beam ion desorption mass spectrometry revealed that Rec forms stable tetramers, which are further stabilized upon RNA binding. The RNA protein complex consists of three Rec tetramers, which bind to multiple sites on the RcRE-preferentially to purine-rich motifs-which represent several low-affinity binding sites. Mutated RcREs, with one to three purine-rich motifs deleted, were still bound and exported by Rec, indicating that the complex folded structure of the RcRE is important for Rec binding. This suggests a binding model where up to three Rec tetramers bind to the complex folded structure of the RcRE and the binding seems to be tightened by recognition of the purine-rich motifs.

HnRNP C Tetramer Measures RNA Length to Classify RNA Polymerase II Transcripts for Export

Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of polyA+ RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

Uncoupling of hTREX demonstrates that UAP56 and hTHO-complex recruitment onto herpesvirus saimiri intronless transcripts is required for replication

Herpesvirus saimiri (HVS) ORF57 nucleocytoplasmic shuttle protein binds viral RNA and interacts with the cellular nuclear export adaptor protein, Aly, to access the TAP-mediated nuclear export pathway. This enables the efficient nuclear export of HVS intronless mRNAs. Herein, we extend these studies and demonstrate that ORF57 recruits several members of hTREX, namely Aly, UAP56 and hTHO-complex proteins, onto the viral mRNAs to assemble an export-competent ribonucleoprotein particle. Moreover, using a transdominant form of Aly which inhibits UAP56 and hTHO-complex association with viral intronless mRNA, we show that complete hTREX recruitment is required for efficient HVS mRNA nuclear export and replication.

The Cellular RNA Export Receptor TAP/NXF1 Is Required for ICP27-Mediated Export of Herpes Simplex Virus 1 RNA, but the TREX Complex Adaptor Protein Aly/REF Appears To Be Dispensable

Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

ICP27 Recruits Aly/REF but Not TAP/NXF1 to Herpes Simplex Virus Type 1 Transcription Sites although TAP/NXF1 Is Required for ICP27 Export

Herpes simplex virus type 1 (HSV-1) protein ICP27 interacts with the cellular export adaptor protein Aly/REF, which is part of the exon junction complex implicated in cellular mRNA export. We previously reported that Aly/REF was no longer associated with splicing factor SC35 sites during infection but instead colocalized with ICP27 in distinct structures. Here we show that these structures colocalize with ICP4 and are sites of HSV-1 transcription. ICP27 mutants with lesions in the region required for the interaction with Aly/REF failed to recruit Aly/REF to viral transcription sites; however, ICP27 export to the cytoplasm was unimpaired, indicating that the interaction of ICP27 with Aly/REF is not required for ICP27 shuttling. ICP27 has also been shown to interact with the cellular mRNA export receptor TAP/NXF1. We report that ICP27 interacts directly with TAP/NXF1 and does not require Aly/REF to bridge the interaction. The C terminus of ICP27 is required; however, the N-terminal leucine-rich region also contributes to the interaction of ICP27 with TAP/NXF1. In contrast to the results found for Aly/REF, mutants that failed to interact with TAP/NXF1 were not exported to the cytoplasm, and TAP/NXF1 was not recruited to sites of HSV-1 transcription. Therefore, the interaction of ICP27 with TAP/NXF1 occurs after ICP27 leaves viral transcription sites. We conclude that ICP27 and the viral RNAs to which it binds are exported via the TAP/NXF1 export receptor.