10 mM Tris–HCl, pH 7.5, 3.3 mM MgCl2 and 250 mM sucrose. After centrifugation, the nuclei were suspended in a solution containing 10 mM MgCl2 and 250 mM sucrose, and further puriﬁed by centrifugation for 10 min at 500g on a sucrose cushion (0.5 mM MgCl2, 350 mM sucrose). The pellet was then lyzed in 10 volumes of 50 mM Na acetate, pH 5.1, 140 mM NaCl, 0.3% SDS and the nuclear RNAs were extracted twice with phenol. Total and cytoplasmic RNAs were puriﬁed with Trizol reagent. After alcohol precipitation, RNA pellets were dissolved in H2O, quantiﬁed using a Nanodrop spectro- photometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA) and diluted to 1 mg/ml. Analysis of ribosomes by sucrose density gradient centrifugation Forty-eight hours after transfection, HeLa cells were treated with 100 mg/ml cycloheximide (Sigma) for 10 min. The cytoplasmic fractions were prepared on ice as described above, except that cycloheximide was added to all buffers. On each 10–50% (w/w) sucrose gradient, 1 mg proteins was loaded, prepared with a Gradient Master former (BioComp Instruments, Fredericton, NB, Canada). The tubes were centrifuged at 4 C and at 36 000 r.p.m. for 105 min in a SW41 rotor (Optima L100XP ultracentrifuge; Beckman Coulter, Villepinte, France). The gradient fractions were collected at OD254 nm with a Foxy Jr. gradient collector (Teledyne Isco, Lincoln, NE, USA). Analysis of pre-rRNA species Pre-rRNA species were analyzed by northern blot. RNAs (3 mg/well for total and nuclear extracts, 6 mg/well for cyto- plasmic extracts), mixed with 3 volumes of loading dye (25 mM MOPS, 6% formaldehyde, 1.5% formamide, 0.16 mg/ml BET) were separated on a 1% agarose gel in the presence of MOPS buffer (20 mM MOPS, 6 mM sodium acetate, 7 10 2 N NaOH, 0.5 M EDTA pH 8.0) containing 6% formaldehyde, and run in MOPS/for- maldehyde buffer (1 MOPS, 6% formaldehyde) at 140 V. RNAs were then transferred to a Hybond N+ nylon membrane (GE Healthcare, Orsay, France). After ﬁxation by UV-crosslinking, the membranes were pre-hybridized for 1 h at 45 C in 6 SSC, 5 Denhardt’s solution, 0.5% SDS, 0.9 mg/ml tRNA. The 32P-labeled oligodeoxy- nucleotide probe was then added and incubated overnight at 45 C. The probes used in the present study were 18S (50-tttacttcctctagatagtcaagttcgacc-30), 50ITS1 (50-cctcgccctc cgggctccgttaatgatc-30), ITS1-160 (50-tctccctcccgagttctc gg-30), ITS1-605 (50-ttaaacctccgcgccggaac-30), ITS1-639 (50-gacccacgggcggacgggcgatc-30), ITS1-721 (50-ggagcggagt ccgcggtg-30), ITS1-779 (50-gtaaagcccccacccgacggc-30), ITS2b (50-ctgcgagggaacccccagccgcgca-30), ITS2d/e (50-gcg cgacggcggacgacaccgcggcgtc-30) and 28S (50-cccgttccctt ggctgtggtttcgctagata-30). For detection of ITS2, probes ITS2b and ITS2d/e were mixed in equal amounts. After hybridization, the membranes were washed twice for 10 min at room temperature in 2 SSC, SDS 0.1% and once in 1 SSC, SDS 0.1%. Labeled RNA signals were acquired with a FLA2000 PhosporImager Nucleic Acids Research, 2011, Vol. 39, No. 1 283 (Fuji, Stamford, CT, USA) and quantiﬁed with ImageGauge software. The 30RACE analysis was adapted from Kiss and Filipowicz (41). The forward primer used for PCR ampliﬁcation spanned nucleotides 582–599 in the ITS1: the ITS1-583 primer (50-ccgccgtccaggtaccta-30). The ampliﬁed fragments were subcloned and automatically sequenced. Detection of ﬂuorescent proteins and ﬂuorescence in situ hybridization microscopy For ﬂuorescence in situ hybridization (FISH) experiments, cells grown on glass cover slips were washed twice in PBS and then ﬁxed for 30 min with 4% paraformaldehyde in PBS (EMS, Hartﬁeld, PA, USA). After being rinsed in PBS, cells were permeabilized for 18 h in 70% ethanol at 4 C. After two washes in 2 SSC containing 10% formamide, hybridization was performed in the dark at 37 C for 5 h in a buffer containing 10% formamide, 2 SSC, 0.5 mg/ml tRNA, 10% dextran sulfate, 250 mg/ ml BSA, 10 mM ribonucleoside vanadyl complexes and 0.5 ng/ml of each probe. Precursors to the 18S rRNA were localized with the 50-ITS1 probe conjugated to Cy3 or Cy5 (GE Healthcare) on amino-modiﬁed deoxy- thymidine (a probe complementary to the 30-end of the 18S rRNA and the 50-end of the ITS1), with the ETS1-b probe (50-agacgacaacgcctgacacgcacggcac-30) conjugated to Cy5 (localized between the 50-end of the ETS1 and the 01/A0 cleavage site), with the ETS1-1399 probe (50-cgctagagaaggcttttctc-30) conjugated to Cy5 (which hybridizes to the 47S pre-rRNA between 01/A0 and A0 cleavage sites) or with the 5.8S-ITS2 probe (50-gcgattgatcggcaagcgacgctc-30) conjugated to Cy3 (a probe complementary to the 30-end of the 5.8S rRNA and the 50-end of the ITS2). After two washes at 37 C with 2 SSC containing 10% formamide, the cover slips were rinsed in PBS and DNA was counterstained with 1 mg/ml Hoechst 33342 (Molecular Probes). After 10 min incuba- tion at room temperature, the cover slips were rinsed twice in PBS and mounted in Mowiol 4.88 (Polyscience Inc., Eppelheim, Germany). For detection of ﬂuorescent proteins after transient transfection, cells grown on glass cover slips were washed twice in PBS and then ﬁxed for 30 min with 4% paraformaldehyde in PBS (EMS). After being washed twice in PBS, DNA was counterstained with Hoechst 33342 for 10 min at room temperature. The cover slips were then rinsed twice in PBS and mounted in Mowiol 4.88. Observations were made with an inverted IX-81 micro- scope (Olympus, Rungis, France), equipped with a motorized stage (M a¨rzha¨ user, Wetzlar-Steindorf, Germany) and a Coolsnap HQ camera (Photometrics, Tucson, AZ, USA), driven by MetaMorph (Molecular Devices, Downington, PA, USA). Cells were observed using a planapochromat 100, 1.4 numerical aperture oil immersion objective, mounted on a PIFOC piezo stepper (PI, Karlsruhe, Germany). The light source was a Polychrome V monochromator (Till Photonics GmbH, Gr a¨felﬁng, Germany) equipped with a 150 W Xenon source and used with a 15-nm bandwidth. For