282 Nucleic Acids Research, 2011, Vol. 39, No. 1 are also required for the ﬁnal cytoplasmic steps, but their molecular function remains to be elucidated (22,28,29). Finally, Nob1p is the endoribonuclease responsible for the cleavage at site D, which matures the 20S pre-rRNA into the 18S rRNA in the cytoplasm (30–33). Assembly of the pre-ribosomal factors into pre- ribosomes is presumably highly coordinated with pre-rRNA processing and intracellular transport of the particles. Recently, the pre-40S particle composition was characterized in human cells (26). It appears that the pre-ribosomal factors assembled in the pre-40S particle are highly conserved between Saccharomyces cerevisiae and human cells. However, ITS1 processing in yeast and mammals is clearly different. After ITS1 cleavage at site A2 in yeast, elimination of the ITS1 within the pre-40S particles requires a single cytoplasmic cleavage step at the 30-end of the 18S rRNA (Figure 1B) (13). In contrast, one detects two abundant intermediates in pre-40S particles in mammalian cells, namely the 21S and 18S-E pre-rRNAs. These RNAs correspond to two processing steps, the 21S pre-rRNA being cleaved in the nucleus and the 18S-E species in the cytoplasm (15) (Figure 1B). Therefore, even if the protein content of pre-40S particles is conserved, the pre-ribosomal factors must have evolved to accommodate an increase in com- plexity of pre-rRNA maturation from yeast to mammals. To further investigate similarities and differences between yeast and human 40S ribosomal subunit biogen- esis, we have studied the role of the human orthologs of two pre-ribosomal factors implicated at two distinct steps of pre-40S particle processing in yeast: Enp1p and Tsr1p. These factors are both essential in yeast. In addition, they are involved at different steps of pre-40S maturation: Enp1p is required for efﬁcient cleavage of the ITS1 in the nucleus (7), whereas Tsr1p is necessary for cleavage at the 30-end of the 18S rRNA in the cytoplasm (22,28,29). hTsr1, the human ortholog of Tsr1p, was recently found in human pre-40S particles, but its speciﬁc role in 40S subunit production is still unclear (26). Bystin/Enp1, the human ortholog of Enp1p, was ﬁrst identiﬁed as a com- ponent of a cytoplasmic complex-mediating adhesion of human trophoblast and endometrial epithelial cells in vitro (34,35), as well as an essential protein for early embryonic development after implantation of the embryo in mouse and rat (36–38). Recently, it was reported that human bystin is overexpressed in hepatocellular carcinoma (HCC) and is essential for cell growth and tumor devel- opment (39). The involvement of bystin in ribosome bio- genesis has also been described (38,40). Further analysis of the functions of bystin and hTsr1 in human ribosome bio- genesis indicates that these two proteins are indeed func- tional orthologs of their yeast counterparts, but highlights differences in pre-40S particle biogenesis between yeast and human. MATERIALS AND METHODS Plasmids To construct pEGFP-bystin and pEGFP-hTsr1, bystin and hTsr1 cDNAs were generated by RT-PCR and cloned into the HindIII and XbaI sites of pEGFP-C3 (Clontech Laboratories, Mountain View, CA, USA), in-frame with the sequence encoding EGFP. EGFP alone was expressed from pEGFP-C3. Transfection of HeLa cells was performed by electro-transformation with 10 mg of plasmidic DNA, as described below for siRNAs. Cell culture and inhibitor treatments Human cervical carcinoma HeLa cells were grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM) containing Glutamax as a supply of L-glutamine, and supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Paisley, UK). Cells were incubated at 37 C in 5% CO2. RNA polymerase I transcription was select- ively inhibited by 50 ng/ml actinomycin D for 2 h. Knockdown of gene expression with small interfering RNAs Two or three 21-mer siRNA duplexes were designed and purchased from Eurogentec (Seraing, Belgium) to knockdown expression of human genes encoding bystin (GenBank accession number: NM_004053) and hTsr1 (GenBank accession number: NM_018128), which are two ribosomal factors associated with the small 40S subunit. After washing the cells once in DMEM (without serum and antibiotics), 10 ml of 100 mM siRNA solution were added on ice to 107 HeLa cells in suspension in 200 ml of the same medium. Electro-transformation was performed at 250 V and at 950 mF with a Gene Pulser (Bio-Rad, Hercules, CA, USA), in a cuvette with a 4-mm inter-electrode distance. The cell suspension was then plated on a 200-cm2 Petri dish containing 20 ml DMEM supplemented with calf serum and antibiotics. Control samples were electro-transformed with a scramble siRNA (siRNA-negative control duplex; Eurogentec). The efﬁciency of the downregulation induced by each siRNA was assessed by quantitative reverse transcriptase-PCR, taking GAPDH as an internal control to normalize expression of the target genes. As the most efﬁcient knockdowns were obtained with a mixture of siRNAs bystin-1 (50-cugcccaaggcauuu aagadtdt-30) and bystin-2 (50-gagguugagacagucaugud tdt-30), and with siRNA tsr1-1 (50-cuggaacaguacacuugaa dtdt-30), these conditions were used for the experiments herein described. The siRNA targeting hRPS15 (siRNA rps15-2: 50-UCACCUACAAGCCCGUAAA-30) has been described previously (15). Cell fractionation and RNA extractions After treatment with siRNAs for 48 h, the cells were suc- cessively washed at 4 C with DMEM, PBS and Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl). One-tenth of the suspension was kept for total RNA ex- traction. The cells were then mechanically disrupted with a Dounce homogenizer in Buffer A containing 0.5 mM DTT. After centrifugation at 4 C and at 1000g for 10 min, the supernatant (i.e. cytoplasmic fraction) was frozen. The pellet containing the nuclei was washed with