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. 2006 Apr 24;173(2):207-18.
doi: 10.1083/jcb.200601105. Epub 2006 Apr 17.

Stepwise RNP Assembly at the Site of H/ACA RNA Transcription in Human Cells

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Free PMC article

Stepwise RNP Assembly at the Site of H/ACA RNA Transcription in Human Cells

Xavier Darzacq et al. J Cell Biol. .
Free PMC article

Abstract

Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.

Figures

Figure 1.
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 by SDS-PAGE before (I) and after immunoprecipitation (IP). The input lanes (I) contained 10% of the proteins used for IP. In each panel, the precipitated proteins are indicated in bold. The antibodies used for IP were directed against NAP57 (A, D, and E) or the HA epitope (B and C). 15% Tricine SDS-PAGE was used for analysis. The migration position and relative molecular mass of marker proteins is indicated on the left, and the mobility of the translated proteins is marked on the right. For unknown reasons and as previously observed (Wang and Meier, 2004), immunoprecipitated NAP57 migrated anomalously on occasion (vertical bars in B). In some experiments, the amount of cDNA encoding one of the proteins was increased to such an extent that the corresponding translated protein at least doubled in abundance (D and E). Fibrillarin was added as a negative control (C and D). (F) Schematic depiction of the mutually exclusive binding of NAF1 and GAR1 to NAP57 in the context of the core trimer.
Figure 2.
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 RNP components (panel 2). Double fluorescence with antibodies to NAP57 (A), NHP2 (B), GAR1 (C), and by FISH with probes to the H/ACA RNAs E3, ACA8, ACA18, and ACA25 (D). (E) Western blot of whole cell extracts probed with affinity-purified NAF1 peptide antibodies and developed by enhanced chemiluminescence. (F and G) Indirect double immunofluorescence on fixed and permeabilized HeLa cells with anti-NAF1 (panel 1) and anti-fibrillarin (F, panel 2) or anti-coilin antibodies (G, panel 2). The corresponding phase-contrast image is shown in panel 3. Note the absence of NAF1 from nucleoli (fibrillarin stain) and its lack of enrichment in Cajal bodies (coilin stain). (H and I) Heterokaryon assay. Double fluorescence of fixed individual (bottom) and fused cells (outlined) 15 min (H) and 4 h (I) after fusion stained for NAF1 (panel 1) and DNA (DAPI; panel 2). Note that NAF1 antibodies strongly label individual HeLa (asterisks) but not NIH3T3 nuclei (arrows), whereas both types of nuclei stain equally at intermediate levels only 4 h after fusion, indicating equilibration, i.e., shuttling of NAF1 (I, panel 1).
Figure 3.
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 the control of the tetracycline response element (TRE) and was induced in the presence of doxycycline (dox) by the transactivator rtTA. The construct contained the polyadenylation/cleavage signal and transcriptional terminator (term) of the bovine growth hormone. The size of the probe used in the RNase protection assay (E) is indicated underneath. (B) Fluorescent micrographs of an E3 cell induced for transgene expression and assayed by FISH with Cy3-labeled probes to exon 1 and 2 (panel 1) and a Cy5-labeled probe to intron 1 (panel 2) and by direct fluorescence for MS2-GFP (panel 3) and for β-globin–CFP (panel 4). Panel 5 depicts the corresponding differential interference contrast image of the cell. (C and D) Triple fluorescence by FISH with probes to the induced rat E3 snoRNA (panel 1), which cross-hybridizes with the endogenous human E3 in uninduced cells (asterisks), and to exon 1 and 2 for identification of the transcription sites (panel 2), and by direct fluorescence of β-globin–CFP in peroxisomes (panel 3) to differentiate the induced cells from the uninduced cells. Insets (width = 2.6 μm) show a magnification of the transcription sites (arrows). (E) RNase protection assay. Autoradiograph of the sequencing gel used to separate the fragments protected from digestion by RNase A and T1. Yeast tRNA (lane 1) and total RNA from E3 (lanes 2 and 3) or E3-minus cells (lanes 4 and 5) isolated before (lanes 2 and 4) or 24 h after (lanes 3 and 5) induction with doxycycline were hybridized to the radiolabeled probe indicated in A. In addition, a separate probe corresponding to 100 nucleotides of SRP RNA was included in all samples as a control. The migrating positions of the protected fragments are indicated on the right. Note that because the probe corresponded to the rat E3 snoRNA of the integrated constructs, it protected only the smaller fragments of the endogenous human E3 snoRNA, which differed in 13 nucleotides from that of the rat.
Figure 4.
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. Panel 4 depicts the corresponding phase-contrast image outlining, with a box the area containing the transcription site, which is enlarged in the insets. (B–F) Same as A, except transfected with GFP fused to the proteins indicated on the left. H/ACA RNA transcription sites in E and F were detected by FISH with the probe to intron 1. Note the failure of GAR1-GFP and GFP-Nopp140 to colocalize to the transcription site (arrows). The width of the insets corresponds to 2.6 μm.
Figure 5.
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 site by FISH with the probe to intron 1 (panel 2). Panel 3 shows a merge of the two in pseudocolor. Panel 4 depicts the corresponding phase-contrast image outlining, with a box indicating the area containing the transcription site, which is enlarged in the insets. (B–D) Same as in A, except immunostained for the endogenous proteins indicated on the left, and the transcription site was identified by the fluorescence of transfected MS2-RFP (panel 2). (E) Bar diagram of the percentage of cells, with the indicated proteins observed at the site of H/ACA RNA transcription. The following numbers of cells with a transcription site (in parentheses) were counted for each protein: NAP57 (150); NOP10 (101); NHP2 (101); GAR1 (104); and NAF1 (159). Questionable colocalizations were discounted. (F) ChIPs of the GFP-fusion constructs indicated on top after their transient transfection into E3 cells and induction of the transgene. Immunoprecipitations were performed with GFP antibodies. Ethidium bromide stain of DNA amplified by PCR, with primers to the regions of the transgene indicated on the right and separated on agarose gels. Amplifications of the input (I; odd lanes) and the ChIPs (even lanes) are shown. (G) Same as in B and C, except stained for the Cajal body marker protein coilin.
Figure 6.
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 were transfected with fibrillarin siRNA. (C) RNase protection assay as in Fig. 3 E on RNA from E3 cells transfected with siRNAs directed against the mRNAs of the proteins indicated on top and induced for transgene expression. Note that NAP57 and NAF1, but not fibrillarin, siRNAs abolished specifically the accumulation of induced rat E3 snoRNA but not of the exon of the gene from which it was expressed. (D) Same as A, except the cells were transfected with NAF1 siRNA. (E) NAF1 is not present at the H/ACA RNA transcription site in cells knocked down for NAP57. Triple fluorescence of endogenous NAP57 (panel 1), FISH with intron 1 probe marking the transcription site (panel 2), and transfected GFP-NAF1 (panel 3) in cells transfected with NAP57 siRNA. Panel 4 shows a phase-contrast image of the same cells and outlines the area of the inset. The arrow points to an unsilenced cell. The width of inset is 2.6 μm. (F) Western blots of lysates from cells treated with siRNA to fibrillarin (lane 1) and NAF1 (lane 2), probed with antibodies to the antigens indicated on the right, and scanned on an infrared imaging system.
Figure 7.
Figure 7.
Three-step model of human H/ACA RNP assembly. NAF1 associates with NAP57 in the cytoplasm (1). The complex is recruited to the site of H/ACA RNA transcription, as are NOP10 and NHP2 (2). GAR1 replaces NAF1 to yield mature H/ACA RNPs in nucleoli and Cajal bodies (3). Released NAF1 recycles across the nuclear envelope. NOP10 and NHP2 may already bind to NAP57 in the cytoplasm.

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