Bystin in human cancer cells: intracellular localization and function in ribosome biogenesis

Abstract

Although bystin has been identified as a protein potentially involved in embryo implantation (a process unique to mammals) in humans, the bystin gene is evolutionarily conserved from yeast to humans. DNA microarray data indicates that bystin is overexpressed in human cancers, suggesting that it promotes cell growth. We undertook RT (reverse transcription)-PCR and immunoblotting, and confirmed that bystin mRNA and protein respectively are expressed in human cancer cell lines, including HeLa. Subcellular fractionation identified bystin protein as nuclear and cytoplasmic, and immunofluorescence showed that nuclear bystin localizes mainly in the nucleolus. Sucrose gradient ultracentrifugation of total cytoplasmic ribosomes revealed preferential association of bystin with the 40S subunit fractions. To analyse its function, bystin expression in cells was suppressed by RNAi (RNA interference). Pulse-chase analysis of ribosomal RNA processing suggested that bystin knockdown delays processing of 18S ribosomal RNA, a component of the 40S 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.

Figure 1. Expression of bystin in human cell lines

(A) RT–PCR analysis of bystin mRNA. cDNAs were prepared by RT of RNA from the indicated human cell lines, amplified by PCR with primers designed from bystin cDNA, and analysed by electrophoresis. +, with reverse transcriptase; −, without reverse transcriptase. Sizes are indicated in bp. (B) Immunoblot analysis of bystin protein. Lysates of cells indicated were immunoblotted with affinity-purified anti-bystin antibody. A band corresponding to putative bystin protein is indicated by arrow. Sizes are indicated in kDa. (C) Immunoblotting of FLAG-tagged bystin. HeLa cells were transiently transfected with mock DNA (lanes 1) and an expression vector for bystin–FLAG (lanes 2) and immunoblotted with the anti-bystin antibody. The antibody was treated with the immunogenic (immuno.) peptide (right-hand blot) or with control peptide (left-hand blot) before use. Sizes are indicated in kDa. In (B) and (C), proteins from approx. 10⁵ cells were loaded into each well and separated in 10% polyacrylamide gels.

Figure 2. Bystin protein in nuclei, ribosomes and cytoplasmic fractions

HeLa (lanes 1–4) and HEK-293T (lanes 5–8) cells were subjected to subcellular fractionation as indicated (see the Experimental section). Each fraction was analysed by immunoblotting with the antibodies indicated on the right. Markers used were fibrillarin for nuclei (nuc.), F₁F₀ ATP synthase (complex V) β subunit for mitochondria (mito.), ribosomal protein S6 for P100 and α-tubulin for cytoplasmic S100.

Figure 3. Localization of bystin in the nucleolus

(A) HeLa cells were double-immunostained with antibodies against bystin (red) and α-tubulin (green). Note that bystin is detected in large nuclear particles (arrows, left-hand panel). (B) Upper panels: HeLa cells were double-immunostained with antibodies against bystin (red) and the nucleolar marker fibrillarin (green). Bystin immunostaining in merged image overlaps with that of fibrillarin. Lower panels: in contrast, in cells stained for bystin (red) and the nuclear speckle marker SC35 (green), staining does not overlap. (C) Anti-bystin antibody was treated with the immunogenic (immuno.) peptide or with control peptide before use for cell staining. The staining patterns obtained with the anti-bystin antibody disappeared by pre-incubation of the antibody with the immunogenic peptide, but not with the unrelated peptide. DIC, differential interference contrast. Scale bars, 5 μm.

Figure 4. Bystin in ribosomal fractions

Upper panel: total cytoplasmic ribosomes (from lysates of HeLa cells) were fractionated by sucrose density gradient centrifugation, and RNA was detected by absorbance (Abs) at 254 nm. Lower panels: fractions were immunoblotted using the antibodies indicated on the right. Ribosomal proteins L10 and S6 are markers of the 60S and 40S subunits respectively. The input comes from 5% of the unfractionated ribosomes. Note that bystin was detected in fractions with ribosomal 40S subunits, but not those containing polysomes.

Figure 5. Effects of bystin knockdown by RNAi

Down-regulation of bystin inhibits 18S rRNA processing. (A) Effect of siRNA duplexes (50 nM each) identified at the top is shown by immunoblotting for bystin (top) and α-tubulin (bottom). After transfection with siRNAs, cells were incubated in normal medium for 72 h before immunoblotting. Note that siBys-D and siBys-G down-regulate bystin protein, but control siLuc does not. (B) Pulse–chase analysis of rRNA processing. Cells treated for 72 h with siBys-D (lanes 1–4), siBys-G (lanes 9–12), and control siLuc (lanes 5–8 and 13–16) were labelled with [methyl-³H]methionine and chased for 0–60 min. The resulting fluorogram of the gels is shown. Note that putative 21S intermediates (indicated by an arrow on the left) accumulate in siBys-D- and siBys-G-transfected cells but not in control cells. (C) Pathways of rRNA processing in HeLa cells. Some intermediates larger than 28S are omitted. Data taken from [33]. (D) Cell proliferation assay. Cells were treated with the duplexes identified at the bottom and incubated for 96 h, and then cell numbers were counted. Bystin knockdown by siBys-D and siBys-G (black bars) significantly compromised cell viability compared with control siRNA-transfected cells (open bar). siTox (hatched bar) was used as an indicator for successful transfection. Results are means±S.D. (n=3) *P<0.05 (analysed by Student's t test).

Figure 6. Intracellular localization of bystin under nucleolar stress

(A) HeLa cells were treated with (upper panels) or without (lower panels) ActD and stained with antibodies against bystin (red) and the nucleolar marker fibrillarin (green). Note that bystin disappears from fibrillarin-positive nucleoli after ActD treatment [compare upper (+ActD) merged image with lower (−ActD)]. Scale bar, 5 μm. (B) HeLa cells treated with ActD (lanes 1–4) and vehicle (lanes 5–8) were subjected to subcellular fractionation as indicated (see the Experimental section). Each fraction was analysed by immunoblotting with the antibodies indicated on the right. Note that bystin is not detected in the fractions of nuclei or P100, including cytoplasmic ribosomes, after ActD treatment. See the legend to Figure 2 for a definition of markers.

Figure 7. Localization of bystin in HeLa cells under nucleolar and heat stress

(A) HeLa cells transiently overexpressing bystin–FLAG were treated with (upper) and without (lower) ActD and stained with antibodies against bystin (red) and fibrillarin (green). Particles containing bystin in ActD-treated cells are indicated by arrows in merged image (upper panel). (B) Untransfected HeLa cells were incubated at 42 °C for 30 min to induce heat shock. Cells were stained with antibodies to detect endogenous bystin (red) and HSF1 (green). HSF1 is a marker of nuclear stress-induced granules. Nuclear stress-induced granules containing HSF1 are indicated by arrowheads. Note that bystin localization is unchanged under heat-shock conditions. (C) HeLa cells transiently overexpressing bystin–FLAG were treated with ActD and stained with antibodies against bystin (red) and HSF1 (green). Note that the particles containing bystin do not co-localize with HSF1. Scale bars, 5 μm. DIC, differential interference contrast.

Figure 8. Expression of bystin in HeLa and HEK-293T cells before and after treatment with rapamycin

(A) Immunoblotting. HeLa and HEK-293T cells were treated with rapamycin (rap.) and lysed with SDS/PAGE sample buffer. Proteins were separated by SDS/PAGE (10% gels) and subjected to immunodetection using antibodies against the proteins indicated on the left. (B) Measurement of expression levels by using immunoblots shown in (A). Band intensities of the proteins in each treatment were determined by using the image analyser, and are shown relative to that of the control (samples without rapamycin). Results are means±S.D. (n=3). Note that rapamycin partially, but specifically, suppressed the expression of bystin, fibrillarin and ribosomal protein S6 in both cell types (*P<0.05).