We initially undertook heterokaryon-shuttling experiments (Borer et al., 1989
) with the well characterized EBV-transformed suspension cell line, BJAB-B1. Because these cells did not adhere well to glass slides, we switched to the human HKB5cl8 cell line, which is a hybrid between human embryonic kidney 293S (HEK293S) cells and 2B8 cells, which are an EBV-positive Burkitt's lymphoma B-cell line (Cho et al., 2002
; El-Guindy et al., 2002
). HKB5cl8 cells not only attach to the glass slides but are morphologically superior in that the nucleus and cytoplasm can be readily distinguished. By RT-PCR analyses (unpublished data), HKB5cl8 cells establish type I latency (Kieff and Rickinson, 2002
) that is characteristic of Burkitt's lymphoma cells. We also performed Northern blot analyses and found that EBER1 and EBER2 are expressed in HKB5cl8 (, lane 1) at levels only two- to threefold lower than in BJAB-B1 cells (, lane 3).
Figure 1. EBER1 and EBER2 do not shuttle in HKB5cl8 cells. (A) EBER1 and EBER2 expression in HKB5cl8 cells. A Northern blot of 5 μg total RNA from HKB5cl8 (lane 1), BJAB (lane 2), and BJAB-B1 (lane 3) was sequentially probed for EBER1, the U6 loading control, (more ...)
To test whether the endogenously expressed EBERs shuttle in and out of the nucleus, heterokaryons were formed by fusing human HKB5cl8 cells with mouse NIH3T3 cells (Borer et al., 1989
). The human cells had previously been transfected with plasmids expressing the shuttling heterogeneous nuclear ribonucleoprotein (hnRNP) A1-GFP protein (Pinol-Roma and Dreyfuss, 1991
); heterokaryons were identified by the appearance of hnRNP A1-GFP in both the human and the mouse nuclei (, and ). Mouse nuclei were readily distinguished by punctate DAPI staining, which replicates the species-specific nuclear staining difference previously reported for Hoechst dye (Moser et al., 1975
Figure 2. Detection of α2 U1 RNA shuttling. (A) Heterokaryons were prepared as in , except that an α2 U1–expressing plasmid, rather than an hnRNP A1-GFP–expressing plasmid, was transfected into HKB5cl8 cells and no cycloheximide (more ...)
Figure 5. Shuttling of the human La protein is not blocked by LMB. Heterokaryons were made by fusing HEK293 cells transfected with plasmids producing Flag-PP32 (1–3) or Flag-PP32 and hnRNP A1-GFP (4–11) with mouse NIH3T3 cells as described in (more ...)
EBER1 and EBER2 were detected by in situ hybridization using DIG-labeled antisense DNA oligonucleotides. These probes were complementary to the 3′ half of the EBERs, but not to regions including conserved polymerase III promoter elements A and B (which may explain the unique report of cytoplasmic localization of EBERs [Schwemmle et al., 1992
]). As shown in (3–6), EBERs remained in the human nuclei and did not shuttle into the mouse nuclei during the 6-h incubation. HEK293 cells transiently expressing EBERs also did not exhibit shuttling (unpublished data); titration of the EBER-expressing plasmids showed that in situ hybridization signals would have been detected even with RNA levels <10% (as observed by Northern blotting; unpublished data).
To ensure that the nucleocytoplasmic shuttling of RNA, as well as of protein molecules, could be observed in our assays, we examined U1 snRNA. We used a modified human U1 RNA, α2 U1 RNA, in which the first 20 nts are significantly different from either the human or mouse U1 snRNA (Yuo and Weiner, 1989
). This U1 RNA is functional in vivo (Yuo and Weiner, 1989
) and, therefore, is expected to follow the wild-type maturation pathway, which involves export to the cytoplasm before assembly with Sm proteins and reimport into the nucleus (Feeney et al., 1989
; Mattaj et al., 1993
). For heterokaryon assays, we transfected an α2 U1 RNA–expressing plasmid into HKB5cl8 cells and visualized the RNA with probes that hybridize specifically to the modified region. We observed α2 U1 RNA in both the human and the mouse nuclei (, ), indicating that α2 U1 moves out of and back into the nuclei of somatic human cells. Importantly, in the same heterokaryons where U1 shuttling was observed, endogenous EBER1 was confined to the human nuclei (, ); the same result was obtained with a longer 12-h incubation (not depicted), as opposed to a 6-h incubation. In the RNA-shuttling assays, cycloheximide was omitted, ruling out the possibility that the lack of EBER1 shuttling is protein synthesis-dependent. EBER2 was also tested, but we were unable to find a hybridization temperature that would allow simultaneous detection of EBER2 and α2 U1 RNAs (unpublished data).
Figure 3. Lack of oocyte nuclear export and Exp5 binding by EBER1. (A) Oocyte microinjections. A mixture of T7-transcribed, α-[32P]UTP–labeled U6, tRNAPhe, and either wild-type EBER1 or mutant EBER1 lacking its 3′ polyU terminus (0.5–1 (more ...)
The absence of EBER signals from mouse nuclei in heterokaryons could be attributable to the rapid cytoplasmic degradation of RNA once it is exported from the human nucleus. Therefore, we compared the turnover rates of EBER1 and other small RNAs; 7SL and Y1 RNAs are both cytoplasmic and transcribed (like EBERs) by RNA polymerase III, whereas U1 RNA is a nuclear RNA polymerase II product. After the addition of actinomycin D to HKB5cl8 or BJAB-B1 cells, EBER1 exhibited an apparent half-life of 25–30 h (), which is significantly greater than Y1 (apparent half-life of 7 h; Rutjes et al., 1999
) and only slightly less than 7SL and U1 (Fury and Zieve, 1996
). Because shuttling was observed for U1, but not for EBER1 (), and they are both extremely stable RNAs, rapid cytoplasmic degradation cannot explain the lack of EBER1 shuttling.
To confirm nuclear retention in another system, we performed X. laevis oocyte microinjection assays using in vitro–transcribed EBER1, U6, and tRNAPhe. 2.5 h after injection, almost all of the positive nuclear export control, tRNAPhe, was detected in the cytoplasmic fraction (, lanes 1, 4, and 5). In contrast, EBER1 remained in the nucleus, as did the negative export control, U6 RNA (, lanes 1, 4, and 5). To address whether La is responsible for the nuclear retention of EBER1, we repeated the microinjection assays using an EBER1 mutant lacking its 3′ polyU tail (required for stable La binding); the terminal nts were changed from UGUUUUOH to GAACACOH. As expected, this EBER1 mutant exhibits eightfold reduced binding to La, based on immunoprecipitation using BJAB cell extracts (unpublished data). 2.5 h after injection, mutant EBER1 remained in the oocyte nucleus, whereas most tRNAPhe was in the cytoplasm (, lanes 6, 9, and 10). Therefore, it is unlikely that La is responsible for the nuclear retention of EBER1.
Finally, to probe why EBERs are not exported, we performed in vitro exportin 5 (Exp5)–binding assays. Exp5 mediates nuclear export of premicroRNAs and adenovirus noncoding RNA VAI by binding to a terminal stem (Gwizdek et al., 2001
; Brownawell and Macara, 2002
; Yi et al., 2003
; Lund et al., 2004
), which is also proposed to exist in EBER1 (Gwizdek et al., 2001
). Using an electrophoretic mobility shift assay, we performed competition experiments to ask if EBER1 can displace the VARdm RNA (Gwizdek et al., 2003
) from recombinant Exp5. Although unlabeled VARdm efficiently competed with the Exp5-bound substrate (, lanes 2–4), neither EBER1 (, lanes 5–7) nor the negative control U6 RNA (, lanes 8–10) significantly displaced the probe, even at 200-fold excess. The same EBER1 preparation was active in binding its protein ligand L22 (Fok et al., 2006
). Thus, lack of binding to an export receptor may explain why EBER1 is not exported from the nucleus. Moreover, it is unlikely that EBERs function by interfering with host cell microRNA biogenesis, which is consistent with observations (unpublished data) that the level of let-7 microRNA is not altered by the presence of EBERs.
Our strategy in investigating the cellular trafficking of EBERs included testing if its obligatory protein partner La undergoes nucleocytoplasmic shuttling. A typical EBV-infected cell harbors ~5 × 106
copies of each EBER (Lerner et al., 1981
), whereas most human cells express ~2 × 107
molecules of La protein (Wolin and Cedervall, 2002
). Thus, even though EBERs do not shuttle, the La protein could. To examine La protein shuttling, HKB5cl8 cells were transfected with plasmids expressing either the shuttling hnRNP A1-GFP or the nonshuttling hnRNP C1-GFP as controls. After fusion with mouse NIH3T3 cells for 4 h, endogenous human La protein was detected using a monoclonal anti-La antibody that does not cross react with mouse La protein (unpublished data; Wolin, S., personal communication), demonstrated by the lack of nuclear staining of unfused mouse cells, labeled m in (panels 7, 8, 10, and 11) and (panels 1, 2, 4, and 5).
Figure 4. Human La protein undergoes nucleocytoplasmic shuttling in multiple cell lines. Heterokaryons were made by fusing HKB5cl8 (1–6), HeLa (7–9), or HEK293 (10–12) cells, which were transfected with a plasmid producing either the shuttling (more ...)
clearly shows that La shuttled from the human nucleus into the mouse nucleus (, panel 2), mimicking the shuttling of hnRNP A1-GFP in the same heterokaryon (, panel 3). Inclusion of cycloheximide during the fusion period ruled out the possibility that newly synthesized human La protein was imported into mouse nuclei. Although the nonshuttling hnRNP C1-GFP remained in the human nucleus (, panel 6), the human La protein moved into the mouse nucleus (, panel 5). We then confirmed that La nucleocytoplasmic shuttling is not cell-type specific by repeating the experiments with nonvirally infected human cells, HeLa or HEK293. Again, the nonshuttling hnRNP C1-GFP remained in the human nuclei and the human La protein shuttled into the mouse nucleus in both kinds of heterokaryons (, panels 8 and 9 and 11 and 12, respectively). We conclude that La, which is predominantly nuclear in multiple types of mammalian cells (Wolin and Cedervall, 2002
), has the capacity to exit and return to the nucleus.
Next, we asked whether La protein is exported via the Crm1 nuclear export receptor because a human La protein lacking its putative nuclear retention element had been reported to accumulate in the cytoplasm, but to be retained in the nucleus in the presence of the Crm1 inhibitor leptomycin B (LMB; Intine et al., 2002
). To ensure that LMB inhibits Crm1 in heterokaryons of HEK293 cells and NIH3T3 cells, we included as a control PP32, which is a known shuttling protein whose nuclear export is Crm1-dependent (Brennan et al., 2000
). We transfected HEK293 cells with a plasmid-expressing Flag-PP32 and, as expected, observed that both La and Flag-PP32 shuttled from the human to the mouse nucleus (, panels 2 and 3, respectively). In the presence of 30 ng/ml LMB, Flag-PP32, but not La, movement was inhibited (, panels 5, 6, 9, and 10). In this experiment, hnRNP A1-GFP, which does not require Crm1 for nuclear export (Brennan et al., 2000
), was coexpressed to identify the hybrid cells (, panels 7 and 11). Because inhibition of Crm1 blocked the shuttling of Flag-PP32, but not of intact La protein, we conclude that the nuclear export of full-length La is either Crm1 independent or that La is exported by more than one pathway. Further studies are needed to resolve the pathways and whether the phosphorylation state of La regulates its shuttling activity (Intine et al., 2003
Because EBERs do not exit the nucleus of either human cells () or Xenopus laevis
oocytes (; even in the absence of a La binding site), it is not the La protein, but rather some other feature of their RNA structure, that retains the EBERs in the nucleus of EBV-infected cells. We tested the prediction, based on the presence of a terminal stem, that EBERs might bind and interfere with the activity of Exp5 (Gwizdek et al., 2001
), which is limiting in the case of premicroRNA export (Yi et al., 2003
). Our findings suggest that EBERs do not function in this way, but instead participate in some other exclusively nuclear process that enhances the expression of several growth factors, including insulin-like growth factor I, interleukin-9, and interleukin-10 (Kitagawa et al., 2000
; Iwakiri et al., 2003
; Yang et al., 2004
) in EBV-transformed cells. Whether these consequences represent an active function of the EBER particles or arise through partial sequestration of La, ribosomal protein L22, or some other protein partner remains to be determined.