Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis

Abstract

Human aminoacyl-tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl-tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.

Figure 1

Structural arrangement of human MRS and specificity of anti-ARS antibodies. A, MRSs of M. pneumoniae, S. cerevisiae, and H. sapiens are aligned schematically. The core catalytic domain is divided into the NH₂- and COOH-terminal domains (marked with amino acid numbers and dotted lines). The signature sequences for class I ARSs (HIGH and KMSKS; Webster et al. 1984; Hountondji et al. 1986; Ludmerer and Schimmel 1987) are highlighted by bars. Human MRS contains the NH₂- and COOH-terminal extensions that are involved in protein–protein interactions (Rho et al. 1999). An ∼40 aa peptide motif (marked as gray box) homologous to those in other ARSs is present in the COOH-terminal end (Rho et al. 1996, Rho et al. 1998). Two nuclear localization signals (NLS) are present in the COOH-terminal region. B, Polyclonal rabbit antibodies were raised against the purified polypeptides of human MRS, EPRS, RRS, and QRS (see Materials and Methods). The antigenic specificities of the prepared antibodies were determined by immunoblotting of proteins extracted from HeLa cells.

Figure 2

Nucleolar localization of MRS. A, Distributions of four different ARSs in Chang cells were determined by immunostaining using confocal laser scanning microscopy. B, top, Cellular localization of MRS was monitored in different cell lines. Human foreskin fibroblast, HeLa, HpG2, and COS-1 cells were cultivated and stained with anti-MRS as described above. Bar, 10 μm. Bottom, Localization of MRS and nucleolin in Chang cells was determined using anti-MRS rabbit antibody and mouse monoclonal antinucleolin antibody (Santa Cruz) as described in Materials and Methods.

Figure 3

Determination of nuclear and free form MRS. Top, MRS was detected by immunoblotting in the whole cell lysate (WCL), nuclear extract (NCL), and postnuclear supernatant (PNS). Cell fractionation was performed as described previously (Neufeld and White 1997). YY1, lamin B, and Hsp90 were used as nuclear and cytoplasmic markers, respectively. Bottom, The proteins extracted from HeLa cells were fractionated by size exclusion chromatography. The eluted proteins in each fraction were analyzed by immunoblotting with anti-MRS, -p43 (Park et al. 1999), and -EPRS rabbit antibodies.

Figure 4

MRS is translocated to nucleolus upon a mitogenic signal. Top, Cellular localization of MRS was monitored in Chang cells as described in Fig. 2. The nucleolar MRS was apparent in subconfluent (∼70% confluency) cells, but disappeared in confluent (100% confluency) or 5-d serum-starved cells. Bottom, 10% serum or EGF (40 ng/ml), PDGF (40 ng/ml), and insulin (100 μg/ml) were added to 7-d serum-starved cells and the cells were observed 24 h after the treatment. Bar, 10 μm.

Figure 5

Nucleolar localization of MRS depends on rRNA synthesis. Top, HeLa cells grown on glass coverslips were permeabilized in 0.1% Triton X-100 for 5 min at room temperature. The permeabilized cells were incubated with DNase I (0.1 mg/ml) or RNase A (0.1 mg/ml) for 1 h at 37°C and fixed in 10% formaldehyde for 20 min. Digestion of nuclear DNA was confirmed by DAPI staining (data not shown). Bottom, HeLa cells were treated with α-amanitin (2 μg/ml), and α-amanitin (2 μg/ml) + actinomycin D (0.2 μg/ml), for 16 h to inhibit RNA polymerase II and RNA polymerase I + II, respectively. The specific inhibition of RNA polymerase I was performed by incubating HeLa cells with cisplatin (10 μg/ml) for 9 h. The cellular localization of MRS was monitored by immunostaining as described above. Bar, 10 μm.

Figure 6

Nuclear run-on assay for rRNA synthesis. Ribosomal RNA synthesis is blocked with anti-MRS antibody. Top, Synthesis of rRNA was monitored by nuclear run-on assay as described previously (Giraudo et al. 1998). The nuclei isolated from HeLa cells were used for the assay. The synthesized transcripts were hybridized to 18S and 28S rDNAs or cDNA for β-actin on the membrane. The amounts of the hybridized transcripts were quantified by phosphor image analyzer. Bottom, The radioactive intensities of 18S, 28S, and β-actin blots without IgG were taken as 100% and the relative intensities of other blots were shown by percentage. White bars stand for the values of the intensities of 18S and 28S treated with the indicated amounts of mock rabbit IgG. Black, gray, and lined bars represent the relative intensities of the 18S, 28S, and β-actin blots treated with the indicated amounts of anti-MRS antibody, respectively. Similar results were obtained from three independent experiments.

Figure 7

Br-UTP incorporation assay for rRNA synthesis. A, The nuclear rRNA synthesis was carried out in the presence of Br-UTP as described in Materials and Methods. The subconfluent HeLa cells were treated with anti-MRS or mock rabbit IgG in the presence of Br-UTP. The incorporated Br-UTP was then detected with anti-BrdU antibody. Fluorescence nucleolar foci (by RNA polymerase I, arrows) disappeared by the treatment of anti-MRS IgG, but not of mock IgG. Nucleoplasmic foci (by RNA polymerase II) were not affected by either of the two antibodies, confirming the specificity of anti-MRS antibody to the nucleolar rRNA synthesis. B, The nucleolar rRNA synthesis was monitored by immunostaining with anti-Br antibody in the presence of different antibodies and RNA synthesis inhibitors. The nuclear DNA was stained with propidium iodide (PI). The nucleolar rRNA synthesis was blocked with anti-MRS antibody, but not with mock IgG or anti-QRS antibody. The stained foci disappeared with the treatment of cisplatin that inhibits RNA polymerase I, but not with the treatment of α-amanitin that inhibits RNA polymerase II. This confirms that the stained foci resulted from the nucleolar rRNA synthesis. Nucleoplasmic Br-staining is not shown here due to the short exposure.