Human NAF1 and GAR1 bind NAP57 competitively
Because yeast Naf1p interacted with some of the H/ACA core proteins, we tested whether NAF1 did as well and, if so, with which ones (Ito et al., 2001
; Dez et al., 2002
; Fatica et al., 2002
; Ho et al., 2002
; Yang et al., 2002
). For this purpose, we used an in vitro–translation/immunoprecipitation assay that we had previously used to dissect the molecular architecture of mammalian H/ACA RNPs (Wang and Meier, 2004
). Specifically, NAF1 was in vitro translated in the presence of various combinations of H/ACA core proteins (, I lanes). Subsequently, one of the proteins was immunoprecipitated and the precipitates analyzed for coprecipitating proteins (, IP lanes).
Figure 1. Interactions of in vitro–translated and immunoprecipitated H/ACA core proteins and NAF1. Fluorographs of the indicated proteins transcribed/translated from individual cDNAs in rabbit reticulocyte lysate in the presence of [35S]methionine and separated (more ...)
Analysis of the autoradiograms showed that NAF1 efficiently coprecipitated with NAP57 alone (, lane 4) and in the presence of the core trimer (lane 6) but not in the absence of NAP57 (lane 2). Neither NAF1 nor NAP57 alone or together precipitated with HA-NHP2 (, lanes 4, 6, and 8). However, addition of NOP10, which mediates the interaction between NHP2 and NAP57, caused NAP57 (, lane 10) and associated NAF1 to precipitate with HA-NHP2 (lane 12). Although NAP57 efficiently coprecipitated with HA-NOP10 (, lane 4), NAF1 did not (lane 2). However, when NHP2 was included with HA-NOP10, some NAF1 did coprecipitate (, lane 6), suggesting that the association of NHP2 with NOP10 exposed a minor NAF1 interaction domain. As in the case of HA-NHP2, further addition of NAP57 to form the core trimer led to the efficient coprecipitation of NAF1 (, lane 8). In contrast, the box C/D core protein fibrillarin, added as a negative control, was not precipitated in any combination (). Collectively, these findings demonstrated that, similar to its yeast counterpart (Dez et al., 2002
; Fatica et al., 2002
; Yang et al., 2002
), human NAF1 specifically interacted with NAP57 alone or in the context of the core trimer ().
Even in the context of the full complement of core proteins, NAF1 efficiently precipitated with NAP57 (, lane 2). Moreover, when the amount of NAF1 was increased in the input (, compare lanes 1 and 3), the amount of GAR1 relative to that of NHP2 and NOP10 was reduced in the precipitate (, compare lanes 2 and 4). This result indicated that NAF1 competed with GAR1 for NAP57 binding. Indeed, increasing the amount of NAF1 in the input while keeping the amount of the H/ACA core proteins the same (, lanes 1–3) resulted in a concomitant reduction of GAR1 relative to NHP2 and NOP10 in NAP57 precipitates (lanes 4–6). NAF1 may bind more tightly than GAR1 to NAP57 because in this system GAR1 expression was insufficient for converse competition and because of the previously observed inconsistent NAP57 association of GAR1 (Wang and Meier, 2004
). Given the presence of a homologous domain in NAF1 and GAR1 (Fatica et al., 2002
), it is plausible that this domain of each protein binds to the same site in NAP57. Collectively, NAF1 and GAR1 bind NAP57 in the context of the core trimer, but their binding sites overlap (or are identical), precluding simultaneous interaction (). This competition between NAF1 and GAR1 suggests a sequential interaction with NAP57.
NAF1 binds NAP57 in the cell nucleus
Although these biochemical analyses clearly showed that NAF1 interacted with NAP57, it was imperative to ascertain that this interaction occurred in the biological context of the cell nucleus. Toward this end, we developed a novel nuclear tethering assay. For this purpose, NAF1 was fused to the lac repressor, LacI, which binds to lac operator sequences that were stably integrated at a single chromosomal locus of the human U2OS osteosarcoma cell line (2-6-3; Janicki et al., 2004
). Transient transfection of NAF1-LacI into the 2-6-3 U2OS cells resulted in the tethering and concentration of NAF1-LacI at one site in the nucleus, easily detectable by indirect immunofluorescence with anti-NAF1 or -LacI antibodies (, panel 1, arrows). Using double immunofluorescence, we tested what endogenous proteins NAF1-LacI recruited to its tethering site. NAP57 accumulated above its nucleolar levels at the sites of NAF1-LacI concentration, indicating that NAF1 also bound to NAP57 in the nucleus (, panel 2). Similarly, NHP2 was observed at the tethering site, apparently recruited to NAP57 via NOP10 (). Despite its ability to interact with NAP57 (Wang and Meier, 2004
), GAR1 was not concentrated at the nuclear site of the NAP57–NAF1-LacI complex (), consistent with our finding that GAR1 and NAF1 binding to NAP57 was mutually exclusive (). All observed interactions were due to the NAF1 moiety of the LacI fusion protein, as LacI alone failed to recruit any of the proteins tested (unpublished data). These results supported our findings from the in vitro–translation/immunoprecipitation approach ().
Figure 2. In vivo interactions, localization, and shuttling of human NAF1. (A–D) Tethering of transfected NAF1-LacI to lac repressor repeats stably integrated into the genome of U2OS cells (arrows) and double labeled for LacI (panel 1) and endogenous H/ACA (more ...)
Although the absence of GAR1 suggested that NAF1-LacI failed to recruit intact H/ACA RNPs, we confirmed this by probing for the presence of four H/ACA RNAs—E3, ACA8, ACA 18, and ACA25. By using simultaneous FISH detection of the four snoRNAs, no or only negligible amounts of H/ACA RNAs were detected at the sites of NAF1-LacI concentration (). Moreover, Nopp140, which binds mature H/ACA RNPs (Meier and Blobel, 1992
; Yang et al., 2000
; Wang et al., 2002
), failed to be recruited by NAF1-LacI (unpublished data). Therefore, NAF1 in the nucleus interacted with NAP57 alone and/or in the context of the core trimer NAP57–NOP10–NHP2 but not with mature H/ACA RNPs.
Human NAF1 is a shuttling nucleoplasmic protein
Although NAF1-LacI bound to the lac operator sequences in the nucleus, excessive expression led to its accumulation in the cytoplasm (, panel 1). Therefore, to investigate the localization of endogenous NAF1, we raised polyclonal antibodies against a synthetic NAF1 peptide. On Western blots of whole cell extracts, affinity-purified NAF1 antibodies recognized a single protein band that migrated at the same position as in vitro–translated NAF1 (compare and , lane 1). By indirect immunofluorescence, they yielded a nucleoplasmic staining pattern excluding nucleoli (the sites of mature snoRNPs, which were double labeled with antibodies to the C/D snoRNP protein fibrillarin; ). Furthermore, NAF1 was not enriched in Cajal bodies, another site of mature snoRNPs, identified by coilin immunofluorescence ().
Given the nuclear and cytoplasmic localization of the NAF1-LacI fusion protein, we tested whether NAF1 shuttled between the nucleus and cytoplasm using a heterokaryon approach (Borer et al., 1989
). For this purpose, we fused human HeLa and mouse NIH3T3 cells (, outlined cells), whose nuclei can be distinguished by a difference in DNA staining (, panel 2, asterisks and arrows). Because the NAF1 antibodies barely recognized the mouse protein while brightly staining the HeLa nuclei (, panel 1), we could test whether NAF1 equilibrated between the nuclei of different origin in fused cells. To inhibit de novo protein synthesis, the fusions were performed in the presence of cycloheximide. Although NAF1 barely redistributed between the different nuclei right after fusion, it fully equilibrated 4 h later (, compare panel 1 in H and I). Specifically, reduced staining in the HeLa nuclei and increased staining in the 3T3 nuclei of the fused cells indicated equilibration of HeLa NAF1 between the nuclei of different origin, i.e., shuttling of NAF1.
Cell line with inducible and observable H/ACA RNA transcription
So far, our data have indicated that NAF1, although absent from mature H/ACA RNPs, bound NAP57 and the core trimer. Based on the shuttling of NAF1, such an interaction could occur in the cytoplasm followed by nuclear translocation for association with H/ACA RNAs. We reasoned that the most likely sites for nuclear RNP assembly were the sites of H/ACA RNA transcription. To test our hypothesis, we developed a unique cell system. Our four requirements for this system were (1) the inducible transcription of a specific H/ACA RNA, (2) the visualization of its transcription site, (3) the processing of the H/ACA RNA from an intron, and (4) the generation of a functional mRNA. Specifically, two cell lines were generated (based on the U2OS cell line) that had tandem arrays of the following constructs stably integrated at a single locus in their genome. Both constructs consisted of the human β-globin gene with three exons, two introns, and a short 5′ untranslated region (UTR; ; Kiss and Filipowicz, 1995
; Darzacq et al., 2002
). Exon 3 was truncated and fused in-frame to CFP containing the peroxisomal targeting tripeptide Ser-Lys-Leu (SKL) at its COOH terminus (Tsukamoto et al., 2000
); thus, fluorescent peroxisomes resulted from the transcription of a functional mRNA. The 3′ UTR encompassed 18 MS2 stem loop repeats, which provided tight binding sites for the MS2 coat protein when transcribed (Janicki et al., 2004
; Shav-Tal et al., 2004a
), thereby allowing the detection of the transcription locus with a MS2-GFP fusion protein. Most important, the rat H/ACA snoRNA E3 was placed in the second intron of the β-globin gene of the first construct (). Expression of the gene was induced by doxycycline through the transactivator rtTA (reverse tetracycline-responsive transcriptional activator), which was stably integrated in these U2OS cells (). This cell line was termed E3. A control cell line that contained the same construct but without the E3 snoRNA was named E3-minus.
Figure 3. Stable human U2OS cell lines expressing rat H/ACA RNA E3 from an inducible promoter. (A) Schematic of the construct that was stably integrated into the genome of the E3 and E3-minus cell lines. Expression was driven by a minimal CMV promoter (Pmin) under (more ...)
After doxycycline induction of the transgene, proper expression and processing of the RNAs in our cell lines was confirmed by fluorescence microscopy and RNase protection assays. First, using FISH with DNA probes for exon 1 and 2 of the β-globin construct, the exons were detected as a single spot in the nucleoplasm and in mature β-globin–CFP mRNPs in the cytoplasm (, panel 1). Hybridization to the same nucleoplasmic spot with a probe for intron 1 confirmed its identity as a transcription site (, panel 2). Alternatively and for potential live imaging studies, the transcription sites and cytoplasmic mRNPs were detected by transiently transfected MS2-GFP (, panel 3). The presence of the mRNA and its 3′ UTR in the cytoplasm suggested that it was full-length and that the β-globin–CFP pre-mRNA was correctly spliced and exported. This was corroborated by the visualization of its translation product, β-globin–CFP, in peroxisomes where it localized by virtue of its COOH-terminal SKL targeting sequence (, panel 4). Importantly, in the absence of doxycycline and transgene transcription, no transcription sites, mRNPs, or β-globin–CFP were detected (unpublished data). The presence of a single transcription site per nucleus suggested that tandem arrays of our constructs integrated at a single site in the genome of these U2OS cells (Shav-Tal et al., 2004b
; Darzacq et al., 2005
). Indeed, FISH on metaphase spreads corroborated a single insertion site (unpublished data).
In E3 cells, the snoRNA produced from the transgene was detected at the transcription site by FISH with two probes complementary to the rat E3 snoRNA (, panels 1 and 2, arrows and insets). Additionally, E3 accumulated in nucleoli in a granular pattern typical for snoRNPs, suggesting its proper incorporation into H/ACA RNPs (, panel 1). Induced cells were routinely identified by the β-globin–CFP protein product of the transgene in peroxisomes (, panel 3). Because the endogenous human E3 snoRNA differed in 13 nucleotides from the induced rat E3, hybridization to endogenous E3 was weaker (, panel 1, asterisk). In the E3-minus control cell line, no E3 was observed at the transcription site (, panels 1 and 2, arrows and insets) and no enhanced fluorescence occurred in nucleoli of induced versus uninduced cells (, panel 1, asterisk). The transgene, however, was properly expressed as attested by the β-globin–CFP signal in peroxisomes (, panel 3).
As a final quality test of our stable cell lines, we investigated the length of the induced snoRNA and the regulation of gene expression using RNase protection assays. Specifically, a radiolabeled RNA probe was synthesized that spanned the intronic snoRNA and exon 2 of β-globin (). When incubated with total RNA from the cell lines, fragments from this probe hybridizing to the stable target RNAs were protected from digestion by RNase A and T1 and migrated at their expected positions on a sequencing gel (, lanes 2–5). However, hybridization to yeast tRNA yielded no protected fragments (, lane 1). After induction by doxycycline, prominent bands of full-length E3 snoRNA and exon 2 were protected by total RNA from E3 cells (, lane 3) but only exon 2 by RNA from E3-minus cells (lane 5). The generation of full-length E3 snoRNA indicated its proper processing and incorporation into H/ACA RNPs. Full-length exon 2 represented correctly spliced globin-CFP mRNA. Transcription of the transgene was tightly regulated because no E3 and exon 2 were detected in the absence of doxycycline (, lanes 2 and 4). Endogenous E3 snoRNA was present independent of induction of the transgene (, lanes 2–5). Note that only smaller fragments of the probe were protected by partial hybridization to endogenous human E3 snoRNA because of slight sequence differences to the rat E3 of the transgene. As a control, a separate probe was included that protected a 100-nt fragment of the unrelated RNA of the signal recognition particle (SRP). Equal amounts of SRP RNA were protected by total RNA from induced and uninduced cells but not by tRNA (). These data indicated that our transgene was correctly induced, transcribed, and spliced and that the rat E3 snoRNA was properly processed and assembled into stable H/ACA RNPs.
Visualization of the H/ACA core trimer and NAF1 at the site of snoRNA transcription
To test which proteins associated with the nascent H/ACA RNA at its site of transcription, we transfected GFP-tagged constructs into the E3 cell line and induced expression of the transgene. Localization at the transcription site was monitored by double fluorescence with cotransfected MS2-RFP or by FISH with the intron 1 probe (, panel 2). All core proteins localized to nucleoli in their characteristic granular pattern, suggesting their incorporation into H/ACA RNPs (, panel 1). In addition, the proteins of the core trimer, NAP57-, NHP2-, and NOP10-GFP, were identified at the site of H/ACA RNA transcription, indicating an early association of the core trimer with the nascent RNA (, insets). However, the fourth core protein, GAR1-GFP, was consistently absent from H/ACA RNA transcription sites (, arrow and insets). The putative assembly factor, NAF1-GFP, concentrated at the transcription sites but was excluded from nucleoli (). Overexpressed NAF1-GFP accumulated in the cytoplasm like NAF1-LacI ( and , panel 1). These observations suggested an early involvement of NAF1 in H/ACA RNP biogenesis, followed by GAR1 replacement during or after release of the maturing particles from the site of transcription. Indeed, the absence from transcription sites of GFP-Nopp140, a chaperone associated with functional H/ACA RNPs in nucleoli (Wang et al., 2002
), supported the lack of mature particles at the site of transcription (). Additionally, the C/D snoRNP protein GFP-fibrillarin, although localizing properly to nucleoli, was absent from the transcription site (unpublished data). Importantly, none of the proteins was observed at the transcription site of E3-minus cells, confirming that all colocalization was due to the nascent H/ACA RNA.
Figure 4. Colocalization of GFP constructs to the H/ACA RNA transcription site. (A) Double fluorescence of an E3 cell cotransfected with NAP57-GFP (panel 1) and MS2-RFP (panel 2) and induced for transgene expression. Panel 3 shows a merge of the two in pseudocolor. (more ...)
The results with the transfected constructs were corroborated with endogenous proteins by indirect immunofluorescence and quantitated. In the same way as the transiently transfected constructs and in addition to their known location in nucleoli and nucleoplasm, endogenous NAP57, NHP2, and NAF1, but not GAR1, were concentrated at the E3 transcription site (). For quantitation, the presence of each protein, transfected and endogenous, at an E3 transcription site was scored. The identifications of each factor at the site of transcription from at least 100 cells are given as a percentage (). These data documented the presence of NAP57, NOP10, NHP2, and NAF1 at the E3 transcription site but a lack of association of GAR1. The <100% coincidence of the localizing factors with the transcription site was likely caused by variable stages of H/ACA RNA transcription and the fact that uncertain identifications were discounted. Nevertheless, the quantitation supported our conclusions and proved the images in and to be truly representative.
Figure 5. Colocalization of endogenous proteins with the H/ACA RNA transcription site. (A) Double fluorescence of a fixed and permeabilized E3 cell after induction of the transgene and immunostained for endogenous NAP57 (panel 1) and for the H/ACA RNA transcription (more ...)
To confirm the recruitment of NAP57 and NAF1 to the snoRNA transcription site by an independent method, we used ChIP on our induced cell lines transiently transfected with GFP-tagged constructs. Using PCR, we tested for the presence of three DNA fragments in the inputs and ChIPs: the promoter region, the rat snoRNA E3, and the CFP-SKL of the transgene. The presence of the correct fragment in the inputs was confirmed in all cases (, odd lanes). Immunoprecipitation with anti-GFP antibodies detected an association of the positive control, the core histone H2b, with all fragments (, lane 2). Although GAR1 and the control C/D snoRNP protein fibrillarin were not associated with any of the three fragments (, lanes 4 and 6), NAP57 and NAF1 precipitated the fragment corresponding to snoRNA E3 but not the other two (lanes 8 and 10). These results indicated that NAP57 and NAF1 were recruited specifically during transcription of H/ACA snoRNA E3 but not before or after. Altogether, the ChIP data supported our single-cell fluorescence localizations.
For the following three reasons, we also investigated whether Cajal bodies coincided with H/ACA RNA transcription sites. First, Cajal bodies have been implicated as sites of H/ACA RNP–mediated pseudouridylation (Darzacq et al., 2002
). Second, the transcription of other small nuclear RNAs, e.g., U2, had been physically associated with Cajal bodies (Frey et al., 1999
; Smith and Lawrence, 2000
). Third, we wanted to verify that the observed H/ACA RNA transcription sites were unique entities and distinct from Cajal bodies. This was to be expected because GAR1 and Nopp140, both verified Cajal body constituents (Meier and Blobel, 1994
; Pogacic et al., 2000
), were not present at transcription sites (; and ) and because only 27% of our U2OS cells contained Cajal bodies. Indeed, most Cajal bodies, identified by their marker protein coilin, were distinct from the snoRNA E3 transcription site (). Nevertheless, in a small number of instances (18 out of 245 cells with both Cajal bodies and transcription sites), an association of the two was observed, suggesting that, as in the case of U2 snRNA, there may be some relationship between active snoRNA genes and Cajal bodies.
NAF1 is essential for stable H/ACA RNA accumulation and is recruited to the transcription site in a NAP57-dependent fashion
Having established that both NAP57 and NAF1 were recruited to the site of H/ACA RNA transcription, we used a siRNA approach to investigate how the appearance of the two proteins related to each other. For this purpose, E3 cells were transiently transfected with NAP57 siRNA for 72 h and the knockdown confirmed by indirect immunofluorescence. NAP57 was knocked down in the nucleoli of >50% of cells, whereas the levels of fibrillarin in the same nucleoli remained unaffected (, compare panels 1 and 2). Similarly, knockdown of fibrillarin left the amount and distribution of NAP57 unaffected (, compare panels 1 and 2). As judged by comparison of fluorescence intensities of nucleoli in silenced versus unsilenced cells (, arrows), NAP57 and fibrillarin were knocked down by ~60 and ~80%, respectively.
Figure 6. NAP57 and NAF1 RNA interference. (A) Double immunofluorescence of NAP57 (panel 1) and fibrillarin (panel 2) in E3 cells 3 d after transfection with NAP57 siRNA. Note that NAP57 is not knocked down in all cells (arrow). (B) Same as A, except the cells (more ...)
To test the impact of NAP57 and fibrillarin knockdown on the expression of the transgene, E3 cells were transfected with siRNAs and induced with doxycycline before total RNA isolation and RNase protection analysis. In this manner, fibrillarin knockdown had no impact on the expression and stability of the E3 snoRNA and the globin-CFP mRNA (, lane 1). However, NAP57 knockdown abolished production of stable E3 snoRNA but left globin-CFP mRNA induction unaffected (, lane 2). The impact of NAP57 knockdown on endogenous E3 snoRNA was less severe, reflecting the increased half-life of the snoRNA once incorporated into mature RNPs. In separate experiments, knockdown of NAF1 also caused a specific loss of induced E3 snoRNA but not of exon 2 (, lane 3), even though NAF1 was only reduced by ~50% (). These data indicated that reduced levels of NAP57 and NAF1 abolished packaging of newly synthesized H/ACA RNA into stable RNPs.
Because both NAP57 and NAF1 proved essential for H/ACA RNP biogenesis, we tested whether their recruitment to nascent snoRNA was interdependent. For this purpose, NAF1-GFP was transfected into E3 cells knocked down for NAP57 but induced for transgene expression. Surprisingly, in NAP57-silenced cells, NAF1-GFP was never detected at E3 snoRNA transcription sites identified by FISH with the intron 1 probe (). However, in control fibrillarin knocked down cells, NAF1-GFP was observed at snoRNA transcription sites (unpublished data). Therefore, recruitment of NAF1 to nascent H/ACA RNAs was NAP57 dependent.
Finally, we tested whether NAP57 was also dependent on NAF1. Because of the partial knockdown, individual cells reduced in NAF1 were difficult to unambiguously identify by indirect immunofluorescence. Therefore, Western blotting was used to compare the amount of the two proteins in whole cell lysates (). After NAF1 knockdown, NAP57 levels were reduced compared with those of tubulin and of control cells knocked down for fibrillarin (, compare lanes 1 and 2). Thus, NAP57 levels were dependent on NAF1.