Constitutive Transport Element Research Articles

Recombinant origin and interspecies transmission of a HERV-K(HML-2)-related primate retrovirus with a novel RNA transport element.

HERV-K(HML-2), the youngest clade of human endogenous retroviruses (HERVs), includes many intact or nearly intact proviruses, but no replication competent HML-2 proviruses have been identified in humans. HML-2-related proviruses are present in other primates, including rhesus macaques, but the extent and timing of HML-2 activity in macaques remains unclear. We have identified 145 HML-2-like proviruses in rhesus macaques, including a clade of young, rhesus-specific insertions. Age estimates, intact open reading frames, and insertional polymorphism of these insertions are consistent with recent or ongoing infectious activity in macaques. 106 of the proviruses form a clade characterized by an ~750 bp sequence between env and the 3' long terminal repeat (LTR), derived from an ancient recombination with a HERV-K(HML-8)-related virus. This clade is found in Old World monkeys (OWM), but not great apes, suggesting it originated after the ape/OWM split. We identified similar proviruses in white-cheeked gibbons; the gibbon insertions cluster within the OWM recombinant clade, suggesting interspecies transmission from OWM to gibbons. The LTRs of the youngest proviruses have deletions in U3, which disrupt the Rec Response Element (RcRE), required for nuclear export of unspliced viral RNA. We show that the HML-8-derived region functions as a Rec-independent constitutive transport element (CTE), indicating the ancestral Rec-RcRE export system was replaced by a CTE mechanism.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution.

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions.IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor.

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

RNA and lessons from viruses.

RNA and lessons from viruses.

The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA.

The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.

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.

Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns.

Intron retention is one of the least studied forms of alternative splicing. Through the use of retrovirus and other model systems, it was established many years ago that mRNAs with retained introns are subject to restriction both at the level of nucleocytoplasmic export and cytoplasmic expression. It was also demonstrated that specific cis-acting elements in the mRNA could serve to bypass these restrictions. Here we show that one of these elements, the constitutive transport element (CTE), first identified in the retrovirus MPMV and subsequently in the human NXF1 gene, is a highly conserved element. Using GERP analysis, CTEs with strong primary sequence homology, predicted to display identical secondary structure, were identified in NXF genes from >30 mammalian species. CTEs were also identified in the predicted NXF1 genes of zebrafish and coelacanths. The CTE from the zebrafish NXF1 was shown to function efficiently to achieve expression of mRNA with a retained intron in human cells in conjunction with zebrafish Nxf1 and cofactor Nxt proteins. This demonstrates that all essential functional components for expression of mRNA with retained introns have been conserved from fish to man.

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA.

BackgroundAll retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated.ResultsThe mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag–only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis.ConclusionThese experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA

All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis. These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.

Evolutionarily conserved features of the retained intron in alternative transcripts of the <i>nxf1</i> (nuclear export factor) genes in different organisms

One of the features of intron-containing genes of the nxf (nuclear export factor) family in different organisms is the presence of an evolutionarily conserved exon-intron block: exon 110nt-intron-exon 37nt. The intron in this evolutionarily conserved block, which we call a “cassette” intron, can be excised or retained in alternative transcripts of nxf1. It corresponds to intron 10 -11 inthe genes that are orthologous to nxf1 in vertebrates, and intron 5 -6 inthe genes that are orthologous to nxf1 in Drosophilidae. The alignment of sequences of cassette introns in nxf1 genes in vertebrates has revealed four evolutionarily conserved sequences: 1)5’flanking sequence, 2) a region containing СТЕ (constitutive transport element), 3) third conserved sequence, and 4)3’flanking sequence. Introns 5-6 of nxf1 in Drosophilidae have no similar conserved sequences. The results of sequence alignment demonstrate a similarity between cassette introns of nxf1 in Drosophilidae in two poly(A) sequences. The prevalence of Dm nxf1 transcripts containing cassette intron 5-6 under completely spliced transcripts in the heads of adult Drosophila melanogaster suggests a functional importance of transcripts that contain a retained intron. Evolutionary conservation, which in Drosophilidae is evident in the presence of poly(A) sequences in cassette introns of the nxf1 genes, is an adaptive feature: the poly(A) sequences are capable of mimicking the 3’-end of transcripts, promote transport from the nucleus to the cytoplasm, or are involved in NMD control. The ability to form characteristic secondary structures is a common feature of nxf1 cassette introns.

A Cell-Based Method for Screening RNA-Protein Interactions: Identification of Constitutive Transport Element-Interacting Proteins

We have developed a mammalian cell-based screening platform to identify proteins that assemble into RNA-protein complexes. Based on Tat-mediated activation of the HIV LTR, proteins that interact with an RNA target elicit expression of a GFP reporter and are captured by fluorescence activated cell sorting. This “Tat-hybrid” screening platform was used to identify proteins that interact with the Mason Pfizer monkey virus (MPMV) constitutive transport element (CTE), a structured RNA hairpin that mediates the transport of unspliced viral mRNAs from the nucleus to the cytoplasm. Several hnRNP-like proteins, including hnRNP A1, were identified and shown to interact with the CTE with selectivity in the reporter system comparable to Tap, a known CTE-binding protein. In vitro gel shift and pull-down assays showed that hnRNP A1 is able to form a complex with the CTE and Tap and that the RGG domain of hnRNP A1 mediates binding to Tap. These results suggest that hnRNP-like proteins may be part of larger export-competent RNA-protein complexes and that the RGG domains of these proteins play an important role in directing these binding events. The results also demonstrate the utility of the screening platform for identifying and characterizing new components of RNA-protein complexes.

Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE

BackgroundRetroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.ResultsWe show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.ConclusionsExpression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.

Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA

Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.

Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication.

Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP

Messenger RNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and mediate export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical-half of CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating nuclear export of viral genomic RNAs, and more generally, provide insights on cargo RNA recognition by mRNA export receptors.

Enhancing recombinant protein production in human cell lines with a constitutive transport element and mRNA export proteins

Recent research into mRNA maturation processes in the nucleus has identified a number of proteins involved in mRNA transcription, capping, splicing, end processing and export. Among them, the Tap-p15 heterodimer acts as an mRNA export receptor. Tap-p15 is recruited onto fully processed mRNA in the nucleus, which is ready for export to the cytoplasm, through associating with Aly or SR proteins on mRNA, or by directly associating with a constitutive transport element (CTE), an RNA element derived from type D retroviruses. mRNA containing a CTE is exported to the cytoplasm by directly associating with Tap-p15, even in the absence of Tap-recruiting proteins such as Aly or SR proteins on the mRNA. Here, we showed that the use of a CTE enhanced the expression of recombinant protein in human cell lines. The co-expression of reporter proteins and Tap-p15 also enhanced recombinant protein expression. Moreover, the use of a CTE and Tap-p15 synergistically further enhanced the recombinant protein expression. In addition to Tap-p15, several Tap-p15-recruiting proteins, including Aly and SR proteins, enhanced recombinant protein expression, albeit independently of the CTE. The incorporation of a CTE and Tap-p15-recruiting proteins into protein expression system is useful to increase recombinant protein yield in human cells.

The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function

Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3'-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.

A novel SR protein binding site in a cis-regulatory element of HIV-1

We have previously described a cis enhancing sequence (CES) in the gp41 region of the HIV-1 env gene. It could enhance HIV-1 Gag expression via both rev-dependent and CTE (constitutive transport element)-dependent pathways. We identified a functionally important and conserved region in the CES that contained a predicted binding sequence for an SR protein, ASF/SF2. We show here that ASF/SF2 bound to this sequence in an electrophoretic mobility shift assay and that the putative ASF/SF2-binding sequence was required for the enhancement of Gag expression by CES and might play a role in HIV-1 posttranscriptional regulation.