1. Volume 16, Issue 5, Pages 777-787 (December 2004)
PHAX and CRM1 Are Required Sequentially to Transport U3 snoRNA to Nucleoli 
Séverine Boulon, Céline Verheggen, Beata E. Jady, Cyrille Girard, Christina Pescia, Conception Paul, Jason K. Ospina, Tamas Kiss, A.Gregory Matera, Rémy Bordonné, Edouard Bertrand 
Molecular Cell 
Volume 16, Issue 5, Pages (December 2004)
DOI: /j.molcel

2. Figure 1
The m7G Cap and a Box C/D Motif Can Each Target U3 to Cajal Bodies of HeLa Cells
(A–C) Localization of capped and uncapped U3 variants. The indicated U3 variants (red; left) were microinjected in the nucleus of HeLa cells, and their localization was analyzed after 30 min at 37°C. Cajal bodies were stained by immunofluorescence (green; middle). (A) wild-type U3, (B) U3mut6, and (C) U3ΔC′ΔC. Insets depict zooms of Cajal bodies. The schematic indicates the mutations introduced in U3. In the two bottom panels, nucleocytoplasmic transport was blocked by the coinjection of wheat germ agglutinin (WGA).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 U3 Precursors Bind Sequentially CBC, PHAX, and CRM1
(A) HeLa cells were transfected with the rat gene rU3B.7 and the indicated plasmid, and the RNAs were immunoprecipitated and analyzed by RPA with a 3′ end U3 probe. Five bands were detected: the primary transcript (U3-0), 3′-processing intermediates (U3-I, U3-II, and U3-III), and the mature form (U3-m). The shorter form of U3-m, U3-m2, is indicated with a dot (see text). Left, analysis of the cap structure with an anti-TMG antibody (K121). Middle left, immunoprecipitation of CBC-proteinA with IgG beads. Middle right and right, immunoprecipitation of PHAX-GFP and CRM1-GFP with anti-GFP antibodies. I, 10% of the input; P−, pellet obtained with beads alone; P+, pellet obtained with beads coated with the appropriate antibodies.
(B) HeLa cells were transfected with the rU3B.7 gene and RNAs were immunoprecipitated with antibodies against the indicated proteins.
(C) Extracts of Ref52 cells were immunoprecipitated with anti-PHAX antibodies. The pellet was strongly enriched for U3-I, U3-II, and U3-III.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
U3 Cap Is Hypermethylated in the Nucleus of HeLa Cells, and PHAX Is Required for U3 Biogenesis
(A) Hypermethylation of U3 cap in presence of leptomycin B. HeLa cells were labeled with 32P orthophosphate for 150 min in presence or not of leptomycin B. The RNA was extracted and immunoprecipitated with beads alone (T−), anticap antibodies (H20), or anti-TMG antibodies (K121). H20 recognizes both m7G and TMG caps.
(B) Depletion of PHAX by siRNAs inhibits U3 biogenesis. HeLa cells were transfected with the indicated siRNA, and harvested 48 hr later. Left, the levels of PHAX and GAPDH were determined by Western blotting. Middle, equal amount of RNAs were analyzed by Northern blots with probes for SRP, U3, and U1. Right, the levels of RNA were normalized to SRP, and PHAX depleted cells were plotted as percent of control cells. Data presented as mean ± SD.
(C) PHAX over-expression increases the levels of U3. HeLa cells were transiently transfected with expression vectors for LacZ, rU3B.7, and either GFP or PHAX-GFP. 48 hr later, RNAs were extracted and LacZ and rU3B.7 levels were measured by slot-blot (upper left) and RPA (bottom left), respectively. Only U3-III and U3m are shown. Right, levels of U3 were normalized to LacZ and plotted as the ratio of the value obtained with GFP. Data presented as mean ± SD.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
PHAX and CRM1 Are Required Sequentially to Transport U3 to Nucleoli
(A) Anti-PHAX antibodies inhibit the transport of U3 to Cajal bodies and nucleoli. Nuclei of HeLa cells were microinjected with a mixture containing fluorescent U3, and either anti-GST antibodies (“control Ab”), anti-PHAX antibodies (“PHAX Ab”), or anti-PHAX antibodies plus recombinant PHAX protein (“PHAX Ab + PHAX”). The localization of U3 was analyzed after 30 min at 37°C (red; left). The injected antibodies were revealed with a fluorescent secondary antibody (green; middle).
(B) Anti-PHAX antibodies inhibit the transport of U3ΔCΔC′ to Cajal bodies. As in (A), except that U3ΔCΔC′ was coinjected with WGA.
(C) CRM1 is required to transport U3 to nucleoli, but not to Cajal bodies. HeLa cells were treated with 15 nM leptomycin B for 15 min and then injected with fluorescent U3.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 PHAX and CRM1 Are Present in Cajal Bodies
(A) Localization of PHAX and CRM1 in Cajal bodies. PHAX (green; upper left), CRM1 (green; bottom left), and Cajal bodies (red; middle) were revealed by immunofluorescence. The nucleus is shown by a DAPI staining in blue.
(B) U3 overexpression increases the levels of PHAX in Cajal bodies. HeLa cells were transiently transfected with rU3B.7, and 36 hr later, the localization of PHAX was determined by immunofluorescence with anti-PHAX antibodies (green; left), whereas the localization of U3 was determined by in situ hybridization (red; middle left). Cajal bodies were revealed by immunofluorescence with an anticoilin antibody (blue; middle right).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 PHAX Associates with U8, U13, and Telomerase RNA
(A) U8 and U13. Left, RNA from HeLa cells was immunoprecipitated with beads alone, anti-cap, and anti-TMG antibodies, and analyzed by RPA with 3′ end probes specific for human U13 and mouse U8. In the case of U8, the mouse U8 gene was transfected in HeLa cells. On the right, extracts from HeLa cells transfected with GFP or PHAX-GFP, and either the U13 or U8 genes, were immunoprecipitated with anti-GFP antibodies.
(B) Extract from HeLa cells transfected (left) or not (right) with PHAX-GFP were immunoprecipitated with anti-GFP and anti-PHAX antibodies, respectively, and analyzed by RPA with a probe against telomerase RNA. Abbreviations: I, input; S, supernatant; P, pellet.
Molecular Cell  , DOI: ( /j.molcel )