The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway

Abstract

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP

“…Note that experiments in A and B have been performed at different temperatures resulting in different export rates of the H2a⌬389 mRNA. Additionally, it should be noted that nuclear degradation of CTE RNA caused by inhibition of CTE export has been observed by others (Pasquinelli et al 1997). This phenomenon likely accounts for the reduction in total CTE levels in oocytes injected with unlabeled CTE RNA.…”

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

“…The transportin-binding site being located within the CTE-binding domain, we then tested whether transportin binding to TAP could dissociate preformed TAP/ CTE RNA complexes+ This could provide a mechanism for the release of the CTE RNA in the cytoplasm and the recycling of TAP back to the nucleus after one round of export+ Unexpectedly, binding of TAP to the CTE RNA prevented its interaction with transportin as shown in Figure 5A+ In this assay transportin was selected from a total E. coli lysate on immobilized recombinant TAP fragments+ The specificity of the interaction was tested by adding RanQ69L-GTP (Fig+ 5A, lanes 4, 7, and 10)+ The CTE RNA prevented the interaction of transportin with TAP fragment 61-372, whereas it had no effect on TAP fragments to which it could not bind (Fig+ 5A, lanes 6-8)+ Thus, the inhibitory effect of the CTE RNA is exerted through its direct binding to TAP+ If the CTE RNA prevents TAP binding by transportin, then it should inhibit TAP nuclear uptake+ This hypothesis was tested both in vivo, in Xenopus oocytes, and in vitro in permeabilized HeLa cells+ In both systems the presence of the CTE RNA strongly inhibited TAP nuclear import (Fig+ 5B,C), whereas the M36 RNA had no effect+ Thus, binding of TAP to the CTE RNA precludes its interaction with transportin, and inhibits the import driven by the N-terminal NLS+ As a consequence, TAP molecules exporting the CTE RNA may not be imported back to the nucleus, providing a rational for the strong inhibitory effect of the CTE RNA on the export of cellular mRNAs (Pasquinelli et al+, 1997;Saavedra et al+, 1997)+…”

“…To determine the specificity of the interaction between TAP and the selected proteins, we performed in vitro binding assays+ [ 35 S]methionine-labeled putative TAP partners were synthesized in vitro in rabbit reticulocyte lysates and assayed for binding to glutathione agarose beads coated with either GST-TAP, GST alone, or various TAP fragments fused to GST+ Binding of RanBP2, p205, and KIAA0169 was not further investigated+ In preliminary experiments we noticed that some of the selected proteins bound to GST-TAP only in the presence of nuclear extracts+ This was the case for CRM1, importin-b, Nup88, Nup93, and RanGAP1 (data not shown)+ Additional experiments indicate that TAP/CRM1 interaction was bridged by CAN and probably by other nucleoporins present in the column (data not shown)+ Because TAP-mediated export is distinct from the CRM1 export pathway (Pasquinelli et al+, 1997;Saavedra et al+, 1997;Zolotukhin & Felber, 1997;Bogerd et al+, 1998;Otero et al+, 1998), we conclude that TAP/CRM1 interaction is not relevant for TAP function+ However, our results indicate that the two proteins share common binding sites at the NPC (see below)+ Binding of in vitro-synthesized transportin, CAN, Nup98, hGle2, and E1B-AP5 to GST-TAP neither required nor was stimulated by addition of nuclear extracts (Figs+ 3, 4, and 5 and data not shown) suggesting that these interactions may be direct, and thus were characterized further+ When GST pull-down assays were performed with various fragments of TAP, hGle2 interacted with the C-terminal domain of TAP (TAP371-619) but not with its N-terminal domain (TAP1-372) (Fig+ 3A, lanes 3-5)+ The C-terminal domain of TAP also interacted with all nucleoporins analyzed in this study (Fig+ 7)+ In contrast, E1B-AP5 associates with the N-terminus (TAP1-372) but not with the C-terminal domain of TAP (TAP371-619; Fig+ 3B, lanes 4 and 5)+ Shorter N-terminal TAP fragments (TAP1-265 and TAP1-185) exhibited a reduced binding affinity for E1B-AP5 (Fig+ 3B, lanes 6 and 7), whereas deletion of TAP's first 60 amino acids severely impaired its binding to E1B-AP5 (TAP61-372; Fig+ 3B, lane 8)+ This observation was supported further by the GST pull-down assay shown in Figure 3C+ In this assay, various TAP deletion mutants synthesized in vitro were tested for their ability to bind to an E1B-AP5 fragment expressed in E. coli as a GST fusion+ This E1B-AP5 fragment (101-619) binds TAP with the same efficiency as the full-length protein (data not shown)+ Deletion of TAP residues 22-55 severely impaired its binding to E1B-AP5 (Fig+ 3C, lanes 4-6), whereas short deletions downstream of position 55 had no effect+ Together, our results indicate that fragment 1-372 represents the E1B-AP5 binding domain of TAP+ Within this domain, the first 55 amino acids appear to contribute substantially to the affinity of the interaction+ Although we can- not rule out that the interaction of TAP with hGle2 and E1B-AP5 is mediated by RNA or by a factor present in the reticulocyte lysate, we consider this possibility unlikely as these interactions were neither prevented by microccocal nuclease treatment nor stimulated by including increasing amounts of HeLa extracts, reticulocyte lysate, or RNA in the binding reactions (data not shown and Fig+ 3D)+ Because the E1B-AP5-binding domain of TAP overlaps with its CTE-binding domain, we next tested the effect of the CTE RNA on TAP/E1B-AP5 interaction+ Full-length TAP and an alanine scan mutant (TAP295) that no longer interacts with the CTE RNA …”

Structural and functional analysis of the avian leukemia virus constitutive transport element

“…It is now known that retroviruses have developed at least two distinct mechanisms to permit the nuclear export of the unspliced and incompletely spliced RNAs that are otherwise subject to nuclear retention by cellular splicing commitment factors (Cullen, 1998;Pollard & Malim, 1998;Stutz & Rosbash, 1998)+ The best understood mechanism is the one utilized by human immunodeficiency virus type 1 (HIV-1) and several other complex retroviruses+ HIV-1 encodes a regulatory protein, termed Rev, that binds to, and multimerizes on, a structured RNA target site termed the Rev Response Element (RRE) found in incompletely spliced viral RNAs+ Rev contains a nuclear export signal (NES) that serves to recruit a cellular export factor termed Crm1 to the resultant ribonucleoprotein complex (Fornerod et al+, 1997a;Neville et al+, 1997;Stade et al+, 1997)+ Crm1 in turn targets these viral mRNAs to the nuclear pores and hence to the cytoplasm+ A second, distinct nuclear RNA export pathway is utilized by Mason-Pfizer Monkey Virus (MPMV) (Bray et al+, 1994)+ MPMV is a simple retrovirus and therefore does not encode a regulatory protein comparable to Rev+ Nevertheless, MPMV faces the same dilemma of how to move its genomic RNA from the nucleus to the cytoplasm+ It is now apparent that MPMV also encodes a cis-acting structured RNA target site, in this case termed the constitutive transport element (CTE), that serves as the binding site for a host cell nuclear export factor termed Tap (Grüter et al+, 1998;Braun et al+, 1999;)+ Tap appears able to target CTE-containing viral RNAs to nuclear pores, and hence to the cytoplasm, and therefore serves a role somewhat similar to Crm1 in HIV-1 RNA export (Katahira et al+, 1999)+ However, Crm1 and Tap clearly access distinct nuclear export pathways and appear to act via quite different mechanisms (Pasquinelli et al+, 1997;Saavedra et al+, 1997;+ Although the use of retroviruses as a model system for the identification and study of novel nuclear RNA export pathways has therefore already facilitated the identification of the host cell nuclear export factors Tap and Crm1, it remains possible that retroviruses distinct from HIV-1 and MPMV may utilize yet other RNA export pathways+ In fact, with the exception of MPMV and the other simian type-D retroviruses, little is known about how simple retroviruses export their genomic RNA to the cytoplasm+ However, in at least one other case (i+e+, the avian sarcoma/leukemia viruses), an RNA sequence that appears functionally analogous to the MPMV CTE has been identified (Ogert et al+, 1996)+ Avian sarcoma viruses (ASV) differ from avian leukemia viruses (ALV) because of the presence of the src gene acquired by recombination with host sequences+ The src gene is located between the env gene and the 39 LTR and, during recombination, the noncoding region present between env and the 39 long terminal repeat (LTR) in ALV has been duplicated+ This sequence is therefore referred to as the direct repeat (DR), where DR1 is located 59 to src and DR2 39 to src+ Of course, in the A...…”