The shuttling SR protein 9G8 plays a role in translation of unspliced mRNA containing a constitutive transport element

The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation.     

NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export

Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)⁺ RNA export and in vivo poly (A)⁺ RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains.     

Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling.

Mammalian Tap-p15 and yeast Mex67p-Mtr2p are conserved and essential mRNA export factor complexes that transport mRNPs through the nuclear pore. Here, we report that the small subunit p15 affects the binding of the large subunit Tap to repeat nucleoporins. BIAcore measurements revealed that recombinant Tap binds with high affinity (K(d) in the nm range) to repeat nucleoporins and dissociates from them very slowly. In contrast, when recombinant Tap was bound to p15, the derived heterodimeric complex exhibited a significant lower affinity to FG-repeat nucleoporins (K(d) in the microm range). Furthermore, when recombinant Tap lacking the N-terminal nuclear localization sequences (TapDeltaNLS) was microinjected in mammalian cells, it did not shuttle; however, TapDeltaNLS with bound p15 efficiently shuttles between nucleus and cytoplasm. We conclude that heterodimerization of Tap and p15 is required for shuttling of the functional Tap-p15 mRNA exporter complex.     

Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.     

Mapping the functional domains of HAP95, a protein that binds RNA helicase A and activates the constitutive transport element of type D retroviruses

The complex retroviruses such as human immunodeficiency virus, type 1, employ a virally encoded protein, Rev, to mediate the nuclear export of unspliced and partially spliced mRNA. In contrast, the simian type D retroviruses act through a cis-acting constitutive transport element (CTE) that presumably interacts directly with cellular export proteins. We first reported that RNA helicase A (RHA) is a shuttle protein that binds to functional CTE in vitro and in vivo. Recently, we isolated a novel protein, HAP95, that specifically binds to the nuclear transport domain of RHA and up-regulates CTE-mediated gene expression. Here, using truncation and deletion mutations, we mapped the domains of HAP95 that are important for RHA binding, transactivation of CTE, and nuclear cytoplasmic shuttling. We report evidence for a novel nuclear export signal in HAP95 and showed that the domains involved in RHA binding and nuclear localization are required for CTE activation. Finally, we showed that HAP95 synergizes significantly with RHA on CTE-mediated reporter gene expression and promotes nuclear export of unspliced mRNA in transfected cells. Taken together, these data support the proposal that HAP95 specifically facilitates CTE-mediated gene expression by directly binding to RHA.     

An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.     

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.     

Regulation of HIV-1 env mRNA translation by Rev protein

We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.     

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.