As shown in Figure 1(A), bystin expression was confirmed by RTCPCR. 40S subunit fractions. To analyse its function, bystin expression in cells was suppressed by RNAi (RNA interference). PulseCchase analysis of ribosomal RNA processing suggested that bystin knockdown delays processing of 18S ribosomal RNA, a component of the 40S Clorobiocin subunit. Furthermore, this knockdown significantly inhibited cell proliferation. Our findings suggest that bystin may promote cell proliferation by facilitating ribosome biogenesis, specifically in the production of the 40S subunit. Localization of bystin to the nucleolus, the site of ribosome biogenesis, was blocked by low concentrations of actinomycin D, a reagent that causes nucleolar stress. When bystin was transiently overexpressed in HeLa cells subjected to nucleolar stress, nuclear bystin was included in particles different from the nuclear stress granules induced by heat shock. In contrast, cytoplasmic bystin was barely affected by nucleolar stress. These results suggest that, while bystin may play multiple roles in mammalian cells, a conserved function is to facilitate ribosome biogenesis required for cell growth. and budding yeast ([9] and [10] respectively. Because the amino acid sequence similarity of the human, and yeast protein products is very high [11], fly Bys and yeast Enp1 proteins are considered to be orthologues of mammalian bystin. Bys shows a dynamic expression pattern compatible with a role in cellCcell interaction and proliferation [9]. Both human bystin and fly Bys c-Raf are targets of the growth-regulating transcription factor, Myc [9,12]. Enp1 has been identified as an essential nuclear protein in yeast [10]. A temperature-sensitive gene in mouse results in embryonic lethality shortly after implantation [15]. These results collectively suggest that bystin plays a universal role in cell proliferation and that, in higher organisms, it has additional functions, some of which may be related to cell adhesion. Recent DNA microarray data have revealed the expression patterns of bystin in multiple human cells and tissues (probe name for bystin, 203612_at; LSBM database, http://www.lsbm.org/site_e/database/index.html). A publicly available database shows that levels of bystin Clorobiocin mRNA are relatively low in normal human tissues, consistent with a previous report [7], but expression of the bystin gene increases in cancer cells in various tumour types. Other microarrays analysing surgical specimens of breast tumours have identified bystin in the proliferation cluster [16]. These observations prompted us to investigate bystin’s role in proliferation of cancer cells. In the present study, we show that bystin in human cancer cells plays a role in ribosomal biogenesis, specifically in the processing of 18S rRNA to produce the 40S subunit. EXPERIMENTAL Antibodies Polyclonal anti-bystin antibody was raised in rabbits against a synthetic peptide, MEKLTEKQTEVETVC (corresponding to human bystin amino acid residues 152C165) conjugated to KLH (keyhole-limpet haemocyanin) for immunization (the cysteine added for conjugation Clorobiocin is underlined) [15]. For affinity purification, rabbit antiserum was absorbed on the antigen peptide linked to agarose beads prepared using SulfoLink coupling gel (Pierce Biotechnologies), eluted with 0.2?M glycine/HCl, pH?2.4, and immediately neutralized with 1?M Tris/HCl, pH?8.5. The following antibodies were purchased: mouse monoclonal anti-FLAG tag antibody (M2) and anti–tubulin antibody from Sigma; mouse monoclonal anti-[F1F0 ATP synthase (complex V) subunit] antibody from MitoSciences; mouse monoclonal anti-fibrillarin antibody from EnCor Biotechnology; rat monoclonal anti-HSF1 (heat-shock factor 1) antibody from Upstate; rabbit polyclonal anti-ribosomal protein L10/QM antibody (C-17) from Santa Cruz Biotechnology; rabbit polyclonal anti-(ribosomal protein S6) antibody and anti-[phospho-S6 ribosomal protein (Ser240/Ser244)] antibody from Cell Signaling Technology; and mouse monoclonal anti-SC35 antibody from BD Biosciences. Cell culture Human cell lines of HeLa (cervical carcinoma), Jurkat (T-cell leukaemia), MCF-7 (breast carcinoma), U-937 (monoblastic leukaemia), YMB-1 (breast carcinoma) and HEK-293T (human embryonic kidney) were cultured at 37?C as described [8,17]. For nucleolar stress experiments, HeLa cells were treated with 10?ng/ml (8.0?nM) ActD (actinomycin D) (Sigma) (from a stock solution of 1 1.0?mg/ml in DMSO) for 24?h. Control cells were treated with the same volume of vehicle. For heat shock, cells were treated at 42?C for 30?min. For treatment with rapamycin, cells were incubated for 24?h with 20?nM rapamycin (dissolved in DMSO) (LC Laboratories). Controls cells were treated with the same volume of DMSO. Plasmids and transfections An IMAGE cDNA clone (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”BC050645″,”term_id”:”29792063″,”term_text”:”BC050645″BC050645; clone ID 6011459), encoding 50?kDa bystin (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_004053″,”term_id”:”1519315962″,”term_text”:”NM_004053″NM_004053) [15], was identified by searching EST (expressed sequence tag) databases and was obtained from Invitrogen. The open reading frame was amplified by PCR using forward primer 5-TTT-GAATTCGAGAAAAATGCCCAAATTC-3 (EcoRI site underlined) and reverse primer 5-TTTGGTACCTCCACGGTGATGGGAACA-3 (KpnI site underlined) and inserted between EcoRI and KpnI sites of pFLAG vector (Sigma) to overexpress C-terminally FLAG-tagged bystin in mammalian cells. Cells were transfected using Lipofectamine? Plus (Invitrogen) according to the manufacturer’s instructions. RT (reverse transcription)CPCR Isolation of total RNA with TRIzol? reagent (Invitrogen), deoxyribonuclease I (Invitrogen) treatment, and RT using Superscript II and an oligo(dT)12?18 primer (Invitrogen) were described previously [18]. Partial cDNA (177?bp) of human bystin was amplified by PCR from the RT products as.