Proteomic Analysis of Rodent Ribosomes ... - ACS Publications

Proteomic Analysis of Rodent Ribosomes Revealed Heterogeneity Including Ribosomal Proteins L10-like, L22-like 1, and L39-like Yoshihiko Sugihara, Hiroki Honda, Tomoharu Iida, Takuma Morinaga, Shingo Hino, Tetsuya Okajima, Tsukasa Matsuda, and Daita Nadano* Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan Received October 6, 2009

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome. Keywords: targeted proteomics • mammalian ribosome • cytoplasmic ribosome • ribosomal protein • paralogue • nucleolus • polysome • testis

Introduction Since the discovery of the ribosome by electron microscopy,1 much effort has been devoted to clarifying the structure of this large ribonucleoprotein complex that is specialized for translation of RNA into protein. Such studies have culminated in clarification of the X-ray crystal structure of eubacterial and archaeal ribosomes.2,3

proteins in rats and 78 in yeast, and that ribosomal proteins are highly conserved among eukaryotes.

Knowledge of the structure of eukaryotic ribosomes lags behind what has been accomplished for bacteria.4 This is partly due to the relatively complex constituents of eukaryotic ribosomes, which have extra segments of rRNAs and many more additional ribosomal proteins.5 Many researchers have attempted to unravel the complexity of these protein components of eukaryotic ribosomes over the last 40 years. Ribosomes in rat liver and budding yeast have been analyzed intensively;5–8 such studies have demonstrated that there are 79 ribosomal

The loci of genes encoding ribosomal proteins in humans were published in 2001.9 The advent of genomics has revealed the structures of genes encoding ribosomal proteins in mammals, and these are now listed in the Ribosomal Protein Gene Database (http://ribosome.miyazaki-med.ac.jp). More than a dozen genes paralogous to a single ribosomal protein have also been found.10 The overwhelming majority of such paralogous genes are considered to be nonfunctional pseudogenes because of their lack of a long open reading frame caused by accumulated mutations,9,10 supporting the previous notion that, in mammals, one ribosomal protein is encoded by one corresponding gene.11 Nonetheless, it has been shown that at least three of these paralogous genes have putative coding regions and are expressed at the message level.12 Expression of these paralogous genes at the protein level or inclusion of the translation products in the ribosome has been unclear.

* To whom correspondence should be addressed: Daita Nadano, Ph.D., Associate Professor, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan. Phone: +81-52-789-4130. Fax: +81-52-789-4128. E-mail: [email protected].

Proteomic studies of plant ribosomes have revealed high heterogeneity due to multiple ribosomal paralogues, whose expression is developmentally regulated.13,14 Probably as a result of genome-wide gene duplication, 59 out of the 78 ribosomal proteins in budding yeast retain two genetic copies.

10.1021/pr9008964

 2010 American Chemical Society

Journal of Proteome Research 2010, 9, 1351–1366 1351 Published on Web 01/11/2010

research articles These second copies have been shown to play different roles in vivo.15,16 Hence, these paralogues are unlikely to be a simple backup (‘stunt double’), but rather have evolved with special functions. It has been hypothesized that the control of gene expression lies in part with ribosomal heterogeneity, or the ‘customized ribosome’.17,18 Abnormal alteration of protein components in human ribosomes might lead to diseases such as cancer.19,20 As mutant screening and analysis in mammals are not performed as routinely as in species such as Arabidopsis and yeast, a proteomic survey is currently a suitable method to examine ribosomal heterogeneity in mammals. Taken together, the available data suggest that the mammalian ribosomal proteome would provide valuable clues to understanding not only the structure of these proteins, but also their roles in gene expression, organ development, and body homeostasis. In the present study, we analyzed proteins from purified ribosomes of rodents (mainly mice). We show the existence of heterogeneity including three paralogues of ribosomal proteins. One of the them, mouse ribosomal protein L39-like (L39L), is probably a functional counterpart of human ribosomal protein L39L/L39-2.12,21

Methods Antibodies. As the immunogen to prepare antibody against mouse ribosomal protein L39L, a peptide, CWIQMKTGNKIMYN (the cysteine added for conjugation is underlined, see also Figure 6A) was synthesized by Sigma-Genosys (Ishikari, Hokkaido, Japan) and conjugated to keyhole limpet hemocyanin by using Imject maleimide activated mcKLH (Piece, Rockford, IL). Because this peptide was impossible to be dissolved completely in the conjugation buffer indicated by Piece (for example, phosphate-buffered saline, that is, 10 mM Na2HPO4 containing 1.8 mM KH2PO4, 0.14 M NaCl, and 2.7 mM KCl), the conjugation procedure supplied from the manufacturer was modified as follows. The peptide (1.5 mg) was dissolved in 0.5 mL of water, then mixed with 1.6 mg of the activated hemocyanin, which had been reconstituted with the addition of 0.16 mL of water just before use, and gently rotated for 2 h at room temperature. The whole products, including a small amount of insoluble peptides, were transferred into the Mini dialysis kit (cutoff molecular mass, 1 kDa; GE Healthcare, Little Chalfont, U.K.) and dialyzed at 4 °C against 0.3× phosphate-buffered saline. The dialyzed material was suspended immediately before use so that insoluble peptides were uniformly dispersed. Four female mice (6-week-old ddY; Japan SLC, Hamamatsu, Japan) were immunized by intraperitoneal injection of the immunogen (100 µg peptide/mouse) emulsified with Freund’s complete adjuvant (Difco Laboratories, Detroit, MI). Two weeks after the first injection, mice were boosted by injection with the immunogen (40-50 µg peptide/mouse) emulsified with Freund’s incomplete adjuvant (Difco Laboratories), which was repeated two more times. Three weeks after the last injection, blood was collected from individual mice. The animal experiments in this study were performed under the protocols approved by the Animal Research Committee of Nagoya University. Anti-thioredoxin antibody was raised in mice against recombinant thioredoxin produced in bacteria. The following antibodies were purchased: rabbit polyclonal antibodies against ribosomal protein L10/QM and glutathione S-transferase (GST) from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-fibrillarin antibody from EnCor Biotechnology 1352

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Sugihara et al. (Alachua, FL); mouse monoclonal anti-FLAG tag antibody (M2) from Sigma (St Louis, MO); rabbit polyclonal antibody against green fluorescent protein (GFP) from Medical & Biological Laboratories (Nagoya, Japan); and rabbit polyclonal antiribosomal protein S6 antibody from Cell Signaling Technology (Danvers, MA). Vectors for the Expression of Mouse Ribosomal Protein L39L. A cDNA clone (accession no. AA675604), encoding mouse ribosomal protein L39L, was found by searching EST databases and obtained from Invitrogen (Carlsbad, CA). The cDNA including the coding region was amplified by polymerase chain reaction (PCR) and subcloned into the following three kinds of vectors to express L39L protein with the indicated tag in mammalian cells, basically according to our previous work:22 pEGFP-C1 (GFP-tag at the amino terminus of L39L; BD Bioscience Clontech, Palo Alto, CA), pEGFP-N3 (GFP-tag at the carboxyl terminus of L39L; BD Bioscience Clontech), and pCMV-Tag2B (FLAG-tag at the amino terminus of L39L; Stratagene, La Jolla, CA). Mouse L39L cDNA was subcloned also into the pET32-a vector (Novagen, Madison, WI) to express L39L protein fused with thioredoxin and the His tag in bacteria. Rodent Tissues. Tissues except for the mammary gland were collected from male BALB/c mice (7-10 week old, Japan SLC). Details in collection of the mammary gland from female BALB/c mice (8-9 week old, Japan SLC) in midlactation (day 10 of lactation) and postlactation (48 h after weaning) were described previously.23 Male Sprague-Dawley rats (10 week old, Japan SLC) were used for collection of the testis. Isolation of Total Cytoplasmic Ribosomes. Total cytoplasmic ribosomes, including the free 40S and 60S subunits, the 80S monosome, and polysomes, were isolated from the liver and testis according to the published methods24,25 with a slight modification. Tissue was minced, transferred into a PotterElvehjem Teflon-glass homogenizer on ice, and homogenized in 50 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose, 0.1 M KCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 0.5 mg/mL heparin (Sigma), and 10 µg/mL cycloheximide. Cell debris, nuclei, and mitochondria were removed by centrifugation at 12 000g for 10 min at 4 °C, and the supernatant was centrifuged again. The postmitochondrial supernatant was then mixed with 1/10 vol of 10% sodium deoxycholate in the same buffer. The cytoplasmic ribosomes were precipitated by ultracentrifugation through a 1 M sucrose cushion at 100 000g for 17 h at 2 °C. Pellets were washed briefly with 5 mM Tris-HCl, pH 7.5, containing 50 mM KCl and 1.5 mM MgCl2, and suspended in the same buffer. Isolation of total cytoplasmic ribosomes from the mammary gland was modified according to a previous report.26 The concentration of isolated ribosomes was determined by measuring absorbance at 260 nm, as described previously.24 Profiling and Collection of Polysomes. Isolated ribosomes were layered onto a linear gradient of 15-45% sucrose and ultracentrifuged in the SW41Ti rotor (Beckman, Palo Alto, CA) at 37 000 rpm for 3.0 h at 4 °C. Fractions of 0.5 mL volume were separately collected with continuous monitoring at 254 nm. Details of these procedures were described previously.22 For collection of polysomes, polysomal fractions were pooled (from the liver, for example, Supplementary Figure 1), added as the top layer in ultracentrifuge tubes (model no. 355618, Beckman), which had been filled with 3.0 mL of 20 mM TrisHCl, pH 7.5, containing 50% sucrose, 0.1 mM KCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol, and 1 mM phenylmethanesulfonyl fluoride, and ultracentrifuged in the 70.1 Ti rotor (Beckman)

Ribosomal Heterogeneity in Mammals at 308 000g for 15 h at 4 °C. Pellets were washed, dissolved, and subjected to ribosome determination as done for total cytoplasmic ribosomes. Preparation of Ribosomal Subunits from Total Cytoplasmic Ribosomes. Total cytoplasmic ribosomes were dissociated into subunits by incubation for 30 min on ice with puromycin (Sigma) and 0.5 M KCl and then incubation for 5 min at 37 °C.24 After brief centrifugation at 25 °C, supernatant including ribosomal subunits was layered on top of a 10-40% sucrose gradient and ultracentrifuged at 38 000 rpm for 4.0 h at 25 °C in the SW41 rotor. Monitoring at 254 nm and fraction collection were performed as described in polysomal profiling. Extraction of Ribosomal Proteins from Ribosomes. Proteins were extracted from ribosomes by precipitation of rRNA with acetic acid,27 extensively dialyzed at 4 °C against 1.5% acetic acid by using the Mini dialysis kit, and lyophilized. Two-Dimensional Gel Electrophoresis by the RadicalFree and Highly Reducing (RFHR) Method. The apparatus for the RFHR method,27 which is composed of three types of electrophoresis apparatus (0-D, 1-D, and 2-D), was purchased from Nihon-Eido (Tokyo, Japan). The photos of the NihonEido’s apparatus and an experimental flow using this apparatus are shown in a recent review.28 Sample Concentration with the Vertical 0-D Apparatus: Ribosomal proteins extracted from 4-10 mg ribosomes were dissolved in 0.1 mL 8 M urea containing 2.8 mM 2-mercaptoethanol and kept for 30 min at 40 °C. To the protein solution, 20 µL of 8 M urea containing 30% acrylamide and 2.4 µL of 0.6 M KOH, containing 3.7% (v/v) acetic acid and trace amounts of pyronine G and acridine orange, which were used as migration markers, were added. Polyacrylamide gels (8%; thickness, 2 mm; width, 5 mm; length (height), 40 mm, that is, about half of the length of a gel slot), containing 12 mM KOH, 0.074% (v/v) acetic acid, and 8 M urea, were prepared in the vertical 0-D apparatus and subjected to prerun at 25 °C for 60 min at 100 V. The electrode solutions for prerun were 12 mM KOH/0.074% acetic acid for the lower cathode reservoir and 12 mM KOH/0.074% acetic acid, containing 6 M urea and 1% 2-mercaptoethylamine, for the upper anode reservoir. Samples were then applied onto the top of polyacrylamide gels. Electrophoresis was performed at 25 °C for about 20 min at 100 V until each sample was concentrated as a sharp single band in the gel. The electrode solutions for main run were 12 mM KOH/ 0.074% acetic acid for the lower cathode reservoir and 0.2 M glycine/0.16% (v/v) acetic acid, containing 4 M urea and 3.5% cysteine hydroxychloride, for the upper anode reservoir. Gel pieces (length, about 5 mm), containing concentrated proteins and dyes, were cut with a razor and subjected immediately to the next first-dimensional gel electrophoresis with the 1-D apparatus. In the first- and second-dimensional gel electrophoresis, prerun was performed with air cooling at 25 °C, and main run was done at 4 °C (in a cold room with air cooling). First-Dimensional Gel Electrophoresis under Basic Conditions (pH 8.2) with the Horizontal 1-D Apparatus: Polyacrylamide gels (8%; thickness, 2 mm; width, 5 mm; length, 180 mm), containing 0.4 M Tris, 0.5 N boric acid, 8 M urea, and 21.5 mM EDTA, in the 1-D apparatus were subjected to prerun for 16 h at 100 V. The electrode solutions were 0.4 M Tris, containing 0.5 M boric acid, 6 M urea, 21.5 mM EDTA, and 0.5% (v/v) acetic acid, for the cathode reservoir and 0.4 M Tris, containing 0.5 M boric acid, 6 M urea, 21.5 mM EDTA, and 1% 2-mercaptoethylamine, for the anode reservoir. Each gel piece

research articles including ribosomal proteins was inserted in the space which was 8 cm away from the anodal end of the electrophoresis gel and cut out with a spatula. First-dimensional electrophoresis was performed for 17 h at 150 V. Both electrode solutions were same as those used in prerun. Second-Dimensional Gel Electrophoresis under Acidic Conditions (pH 3.6) with the Vertical 2-D Apparatus: Polyacrylamide gels (18%; thickness, 2 mm; width, 160 mm; length (height), 130 mm), containing 48 mM KOH, 5.2% (v/v) acetic acid, and 8 M urea, were prepared in the vertical 2-D apparatus and subjected to prerun for 16 h at 100 V. The electrode solutions were 48 mM KOH containing 5.2% (v/v) acetic acid for the lower cathode reservoir and 48 mM KOH, containing 5.2% (v/v) acetic acid, 6 M urea, and 0.5% 2-mercaptoethylamine, for the upper anode reservoir. Each first-dimensional gel was then placed onto the top of the second-dimensional gel. Electrophoresis was performed for 44-47 h at 100 V. The electrode solution for the lower cathode reservoir was 0.20 M glycine containing 0.16% acetic acid, to which 1/400 vol of 12 N HCl was added before use.27 The electrode solution for the upper anode reservoir was 0.20 M glycine, containing 0.16% acetic acid, 6 M urea, and 3.4% cysteine hydroxychloride. The procedures described above were modified from the Nihon-Eido’s instructions. The supplier’s instructions (written in Japanese) are basically according to the original paper,27 and described in detail in a very recent review.29 Supplementary Figures 2, 3, and 4A show ribosomal proteins separated under the standard conditions of the supplier. According to the supplier’s instructions, electrophoresis of proteins from 0.5-1.0 mg of rodent ribosomes provided good spot separation in the middle region of gel. After electrophoresis under these standard conditions, 62 ribosomal proteins and Rack1 were identified by mass spectrometry (MS) (Supplementary Figure 2). It was also revealed that very small ribosomal proteins (L39, L40, S27a, S29, and S30) were undetectable under the standard conditions, even when a large amount of sample proteins (from 5-10 mg ribosomes) was applied (Supplementary Figure 4A). First- and second-dimensional gel electrophoresis is performed at room temperature under the standard conditions. We found that these very small proteins were reproducibly detected after electrophoresis at 4 °C (Supplementary Figure 4B). Because proteins on gel migrated slowly at 4 °C, the run time of firstand second-dimensional electrophoresis was extended. In addition, ribosomal proteins P1 and P2 easily slipped away from the anodal end of gel in first-dimensional electrophoresis under the standard conditions (compare Figure 1 and Supplementary Figure 2). This is because P1 and P2 polypeptides are not only acidic (calculated isoelectric points of mouse P1 and P2, 4.07 and 4.22, respectively), but also post-translationally phosphorylated.5 To prevent the loss of these two proteins, it was necessary to tune the position where the sample gel block obtained after the 0-D electrophoresis was inserted and the run time of the first-dimensional electrophoresis. In our final protocols, the compositions of electrophoresis gels and electrode solutions were not altered from the supplier’s instructions. Visualization of Proteins Separated by Two-Dimensional Gel Electrophoresis. After electrophoresis, gel was directly immersed in colloidal Coomassie brilliant blue G in acidic solution including trichloroacetic acid,30,31 gently shaken for 5 h at 50 °C, and stored for about 16 h at 4 °C to achieve maximal detection sensitivity. The gel was subsequently rinsed with 25% ammonium sulfate in water for color intensification.32 Methanol or ethanol, which has been suggested to disturb Journal of Proteome Research • Vol. 9, No. 3, 2010 1353

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Figure 1. Electrophoresis of ribosomal proteins from the mouse liver. Proteins from 7 mg of mouse liver polysomes were subjected to two-dimensional electrophoresis using the modified RFHR method. Ribosomal proteins were separated in the first dimension (horizontal) by electrophoresis on 8% polyacrylamide gel at pH 8.2 and then in the second dimension (vertical) by molecular weight sieving on 18% polyacrylamide gel at pH 3.6. Separated proteins were visualized with colloidal Coomassie brilliant blue. Spots were excised and subjected to tryptic digestion. Proteins were identified by MS and database search. Position of each ribosomal protein thus identified is indicated. The region indicated by “M” is magnified and shown in the inset. The position where sample gel was placed in the first-dimensional electrophoresis is indicated by the open triangle. The positive electrode in the second-dimensional electrophoresis is at the top.

detection of ribosomal proteins P1 and P2 on gel,33 was not used during these staining procedures. In-Gel Digestion with Trypsin. Each protein spot was excised from the stained gel with a scalpel, sliced into small pieces (less than 1 mm3), and transferred in low-proteinbinding microfuge tubes (Prokeep, Watson, Tokyo, Japan), where subsequent digestion was performed. The gel pieces were destained by incubating twice in 0.2 mL of acetonitrile/ 0.1 M NaHCO4 (1:1), and then washed four times with 0.2 mL of 10% (v/v) acetic acid/methanol (1:1). This wash was performed at about 15 °C for 10 min for the first three times and at the same temperature for 16 h the fourth time. After preincubation of the gel pieces for 5 min in 0.2 mL of 10 mM NaHCO4, they were mixed with 0.2 mL of acetonitrile and allowed to dry completely by SpeedVac evaporation. Working trypsin solution was prepared by dissolving Trypsin Gold (Promega) with 50 mM acetic acid at the concentration of 1 mg/mL and then diluting it 100 times with 10 mM NaHCO4. To the dried gel pieces, 4 µL of working trypsin solution (10 ng/µL) was added and kept for 15 min on ice. After the addition of 8 µL of 10 mM NaHCO4, the gel pieces were incubated for 2 h at 37 °C, then for 1 h at 30 °C for digestion. After excess solution was removed, 30 µL of 0.2% trifluoroacetic acid/ acetonitrile (1:1) was added to the gel pieces, and they were shaken for 15 min to extract digested peptides. Second extraction was performed for 15 min with 30 µL of 0.2% trifluoroacetic acid/acetonitrile (1:2). All the extracted peptides were trans1354


ferred into a new Prokeep tube, concentrated to about 2 µL by SpeedVac evaporation, and then subjected to MS. Use of 10 mM NaHCO4, instead of 0.1 M NaHCO4, after destaining improved peak intensities in subsequent MS (data not shown), probably due to minimizing interference caused by excess salt. Further cleanup, for example, with ZipTip C18 resin (Millipore)23 was unnecessary under the present conditions. MS and Protein Identification. The extracted peptides from each protein spot were spotted on the µFocus MALDI plate (Hudson Surface Technology, Newark, NJ). Then 33% saturated R-cyano-4-hydroxycinnamic acid (Sigma) in 0.2% trifluoroacetic acid/acetonitrile (1:1) was applied to the spot and air-dried. MS was performed on 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Mass spectra of the tryptic digests were collected by matrix-assisted laser desorption/ionization time-of-flight tandem MS in positive ion reflectron mode. The spectra were calibrated with the 4700 Proteomics Analyzer calibration mixture (Applied Biosystems) and measured over a mass range of 850-4000 Da. Monoisotopic, singly charged (protonated) masses, [M + H]+, from the mass spectra, were then extracted and analyzed using the online Mascot search engine at the Web site (http://www.matrixscience.com) of Matrix Science (London, U.K.). Trypsin autolysis products were excluded. For peptide mass fingerprinting (PMF), protein identifications were performed by the Mascot peptide mass fingerprint search and assigned using the nonredundant database of the National Center for Biotechnology Information

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Ribosomal Heterogeneity in Mammals Table 1. Primers and Products in RT-PCR ribosomal protein

L10 L10L L12 L39 L39L S5

primer pair

5′-TGTGATTGAGGCTCTGCGAA-3′ 5′-ATCAGGAATGAGCCGCTTCT-3′ 5′-TGATCGAGGCCCTACGCC-3′a 5′-CAGGGATGAGGCGCTTGG-3′a 5′-ATGACATTGCCAAGGCTACC-3′ 5′-AAGACCGGTGTCTCATCTGC-3′ 5′-TCTTCTCACAAGACTTTCCGA-3′ 5′-CAGACCCAGCTTCGTTCTC-3′ 5′-CTTCTCACAAGACCTTCAGG-3′a 5′-TTATAGACCCAATTTGGTTCGT-3′a 5′-CTCTGCAGGTCCTGGTGAAT-3′ 5′-TGGCATAGGAATTGGAGGAG-3

product size (bp)

139 135 231 150b 152b 246

a Each nucleotide residue of a paralogue, which is different from that of the corresponding ribosomal protein, is underlined. b The sequences of these products are shown in Supplementary Figure 8.

(NCBI). Propionamide-cysteine residues (fixed modification) and oxidation of methionine residues (variable modification) were taken into account with a peptide tolerance of (0.1-1.2 Da. One missed cleavage was allowed. For protein identification by tandem MS (MS/MS), the 5 or 10 most intense peptides detected in each MS mode were automatically selected for MS/ MS analysis. Combined peptide mass with fragmentation data were searched at the Mascot MS/MS ion search site. The search parameters were the same as for PMF described above, except a MS/MS tolerance of (0.1-0.6 Da was used. Protein identification was considered significant if the Mascot score was higher than that of a random match at P < 0.05. RNA Preparation and Reverse Transcription (RT)-PCR. Preparation of total RNA from tissues using the Trizol reagent (Invitrogen), treatment of isolated RNA with deoxyribonuclease I, RT with Superscript II and oligo(dT)12-18 primer (Invitrogen), and check of successful RT by amplification of β-actin cDNA were performed as described previously.23 Partial cDNAs of mouse ribosomal proteins L10 and L10like (L10L) were amplified by PCR from the RT products. Primer pairs for those proteins are listed in Table 1. Amplification was carried out using rTaq DNA polymerase (Takara, Otsu, Japan) and the following protocol: denaturation for 4 min at 94 °C and 20 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72 °C. Products were separated on 8% nondenaturing polyacrylamide gel and visualized with ethidium bromide.23 Quantitative PCR was performed using the iQ SYBR Green supermix (Bio-Rad, Hercules, CA), in the iCycler iQ real-time PCR detection system (model no. 170-8740) with iCycler software version 3.0A (Bio-Rad). Primer pairs for mouse ribosomal proteins S5, L12, L39, and L39L (Table 1) and for glyceraldehyde-3-phosphate dehydrogenase23 were used for this analysis. Tissue cDNA, primers, and the iQ SYBR Green supermix were incubated at 94 °C for 4.5 min followed by 45 cycles (0.5 min each) of denaturation, annealing, and extension at 95, 60, and 72 °C, respectively. SYBR Green fluorescence was measured at the end of the extension step of each cycle. Immediately after amplification, melt curve analysis (a rise in temperature from 55 to 95 °C at a rate of 0.5 °C/10 s with continuous acquisition of fluorescence decline) was performed to verify the PCR product. To quantify each gene product of interest, standard curves were constructed by using 10-fold serial dilutions of the mammary gland cDNA. Standard curves

were based on the values of threshold cycle (Ct) and generated by the iCycler software. Experimental target quantities were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase. One-Dimensional Gel Electrophoresis of Proteins, Electroblotting, and Immunodetection. Proteins (molecular mass, >10 kDa) were separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto Immobilon membranes (Millipore, Bedford, MA), reacted first with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (BioRad), and detected using the ECL detection system (GE Healthcare Biosciences, Piscataway, NJ) or the Immobilon western HRP substrate (Millipore), basically according to the previous papers.22,34 Immunoblotting of endogenous ribosomal protein L39L was modified as follows because of its very low molecular mass (see also Table 3). Protein samples were applied onto 20% polyacrylamide gels, electrophoresed with high-molarity buffers for better separation of low-molecular-weight peptides,35 and electroblotted in 25 mM Tris, containing 0.19 M glycine and 35% (v/v) methanol. Blocking of the blots was performed using the PVDF blocking reagent for Can-get-signal (Toyobo). Culture of Mammalian Cells, Transfection, and Fluorescence Microscopy. Human embryonic kidney 293T cells were transiently transfected with vectors using a lipofection reagent, HilyMax (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. Total cell lysates were prepared by homogenizing cells with 1× SDS-PAGE sample buffer25 to check protein expression. GFP fusion proteins and GFP were resolved by SDS-PAGE and identified as a band at the expected molecular mass by immunoblotting with anti-GFP antibody. For microscopic observation, 293T cells were cultured on coverslips, fixed for 15 min with 2% paraformaldehyde in phosphate-buffered saline, and treated for 30 min with phosphate-buffered saline including 1% Triton X-100 and 1% bovine serum albumin (fraction V, Sigma). Cells were then treated with primary antibodies and fluorophore-conjugated secondary antibodies as described previously.21 Digital images of stained cells were captured by using a laser-confocal microscope system (LSMS Pascal, Carl Zeiss, Oberkochen, Germany). Production of Mouse Ribosomal Proteins L39 and L39L in Bacterial Cells. The peptide sequence of mouse ribosomal protein L39 is 100% identical to that of human ribosomal protein L39 (see also Figure 6A). Because human L39 cDNA had been subcloned into pCMV-Tag2B previously,21 this cDNA was inserted into the pGEX-KG vector to express recombinant human/mouse L39 protein fused with GST in bacteria. Escherichia coli DH5R cells were transformed with this vector, and GST-L39 fusion protein was produced upon induction with 0.1 mM isopropyl 1-thio-β-D-galactoside. After cell lysis with sonication, the GST fusion protein was purified using glutathione-Sepharose beads (GE Healthcare). Recombinant L39L protein was produced and purified using the pET-32 vector expressing this protein, which was prepared in this study as described above, BL21 (DE3) pLsyS strain (Novagen), and Hisbind resin (Novagen), according to the Novagen’s protocols. GST-L39 and thioredoxin-L39L proteins were resolved by SDSPAGE and identified as a band at the expected molecular mass by staining the gel with Coomassie brilliant blue R-250. In Silico Analysis. The structural information of genes and their products was obtained at the Entrez Gene site of NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez?db)gene). Public Journal of Proteome Research • Vol. 9, No. 3, 2010 1355

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Figure 2. Identification of ribosomal protein L22L1. (A) The region including spots of ribosomal proteins L22 and L22L1 on gel. Proteins from polysomes of the mouse mammary gland in midlactation and the mouse liver were separated by the modified RFHR method and visualized with colloidal Coomassie brilliant blue. The spots of L22 and L22L1 are indicated by the arrowheads and arrows, respectively. (B) Mass spectrum of L22L1 from the corresponding spot after in-gel digestion with trypsin. Data of the numbered peaks were used for identification by Mascot search (Table 2). The m/z peaks in this panel correspond to singly charged peptides (MH+). (C) Amino acid sequence alignment of mouse L22 and L22L1. Numbers on the right indicate positions of the amino acids. Vertical lines represent identity between a corresponding pair of amino acid residues, and colons represent similarity. Peptide fragments corresponding to the L22L1-specific peaks 1-3 and 5 in (B) are indicated by the solid lines, and a fragment corresponding to the peak 4 common between L22 and L22L1 is indicated by the broken line.

DNA microarray databases36 available at http://biogps.gnf.org were used to examine the tissue distribution of gene transcripts 1356


in mice. The molecular mass and isoelectric point of a protein were calculated from a peptide sequence deduced from the

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Ribosomal Heterogeneity in Mammals Table 2. Identification of Ribosomal Protein L22L1 by MS (Figure 2B) identified peptide (PMF) a

peak

m/z obs.

1 2 3 4 5

2832.3733 3089.5078 1307.6833 1085.5518 1408.7172

m/z calc.

delta

missb

start-end

sequence

2832.3508 3089.4883 1307.6946 1085.5730 1408.7198

+0.0226 +0.0195 -0.0113 -0.0212 -0.0026

0 1 0 1 1

16-39 16-41 48-59 88-95 96-107

FHLDLTHPVEDGIFDSGNFEQFLRc,d FHLDLTHPVEDGIFDSGNFEQFLREKc,d TGNLGNVVHIERc,d NNLRDWLRc VVASDKETYELRc,d

a Each experimental m/z value (MH+) was transformed to a relative molecular mass (Mr) by the Mascot program. The Mr values are shown as “m/z obs.” in this table. b One missed cleavage was allowed in a Mascot search. c All the peptide sequences were also identified by MS/MS analysis (Supplementary Table 1). d These four peptide fragments are specific for L22L1 (Figure 2C).

Table 3. Properties of Mouse Ribosomal Proteins and Their Identified Paralogues ribosomal protein

molecular mass (kDa)a

isoelectric pointa

gene locationb

L10 L10L L22 L22L1 L39 L39L

24.60 24.56 14.76 14.47 6.41 6.34

10.51 10.53 9.61 9.86 13.05 12.91

X A7.3 12 C2 4 E2 3 A3 X F3 16 A1

a

Calculated by the Genetyx software.

b

Data from the Entrez Gene

site.

coding region of its cDNA, by using the Genetyx software (version 7.01; Osaka, Japan). Alignment of nucleotide and peptide sequences was performed with the same software.

Results Detection of 78 Ribosomal Proteins and Rack1 on Electrophoresis Gel. The average mammalian ribosomal protein is small (average molecular mass, 18.5 kDa) and very basic (average isoelectric point, 11.05).5 Proteins in rodent ribosomes were separated by RFHR two-dimensional electrophoresis.27–29 This excellent method was developed by Wada,27 based on the Kalshmidt-Wittmann method.33,37 Standard two-dimensional electrophoresis (for example, using O’Farrell’s method) yields multiple spots produced by artificially induced disulfide bridges, which are scarcely produced in RFHR electrophoresis. Seconddimensional electrophoresis at pH 3.6 improves the separation of small basic proteins. The RFHR method was originally developed for the separation of eubacterial ribosomal proteins. Since eukaryotic ribosomes contain extra protein components that have no homologues in prokaryotes,5,8 we modified the procedures to detect ribosomal proteins in rodents (see the Methods section for details). Proteins separated by the modified RFHR method were detected using colloidal Coomassie Brilliant blue G-250, which has been reported to be suitable for sensitive detection of small basic proteins.31 Visualized protein spots were subjected to in-gel digestion with trypsin and identification by PMF followed by MS/MS. Figure 1 shows an electrophoretogram of ribosomal proteins from mouse liver polysomes. The MS data for identified ribosomal proteins are summarized in Supplementary Table 1. Ribosomal protein S6 was detected as unique multiple spots (see also Supplementary Figure 3), as demonstrated using the Kalshmidt-Wittmann method.38 This multiplicity is due to differences in phosphorylation of five seryl residues of this protein.5,39 Rack1/Gnb2l1 (Asc1 in budding yeast), which is a newly identified eukaryotic ribosomal protein located in the small subunit,40 was also identified from an isolated spot. Although the following four

pairs of ribosomal proteins were not separable under our electrophoresis conditions, the proteins were identified by MS from a mixed spot of each pair: L13 and L13a, L23 and S19, L32 and S24, and L36 and L36a. Finally, 78 out of 79 ribosomal proteins and Rack1 were identified. Ribosomal protein L41 was the only one undetected in this study. L41 is the smallest and extremely basic (calculated molecular mass and isoelectric point, 3.5 kDa and 13.5, respectively), and it is very difficult to fix on the gel. In fact, yeast L41 is reportedly difficult to detect on electrophoresis gel,41 as it elutes close to the solvent front, a behavior similar to its orthologous protein, Thx, from Thermus thermophilus.42 Also, the peptide sequence of mouse L41 indicates that only amino acids and dipeptides are generated after trypsin digestion and unsuitable for identification by our MS method. Lack of Obvious Heterogeneity in Electrophoretic Patterns of Ribosomal Proteins from the Liver, Mammary Gland, and Testis. All tissues produce the necessary types and numbers of proteins to accomplish their specific roles. The liver, as suggested by the richness of its polysomal profile (Supplementary Figure 1), has an overall enhanced level of protein synthesis. In the mammary gland, enormous amounts of secretory milk proteins including caseins are synthesized during lactation,43,44 while in the testis, translational regulation specific for spermatogenesis has been reported.45,46 Although the ribosomes in the liver have been analyzed extensively and reviewed by Wool,5 there are few reports on ribosomes in the mammary gland and testis, and involvement of ribosomal heterogeneity in customized protein synthesis in these two tissues has remained unclear. Therefore, we surveyed heterogeneity of ribosomal proteins in the three different tissues employing the proteomic methods described above. For this purpose, we used polysomes purified from the liver and mammary gland of mice. Because the yield of polysomes from mouse testis was very low (see the polysomal profile in Figure 8A), total cytoplasmic ribosomes of mice and polysomes of rats were used as the source of ribosomal proteins for this tissue. The electrophoresis patterns of ribosomal proteins from mammary gland and testis were quite similar to the patterns obtained from the liver (data not shown; the pattern from mammary gland polysomes, which was obtained by the standard RFHR method, is shown in Supplementary Figure 2). This supports the concept of structural conservation among mammalian ribosomes, which has been indicated by electrophoretic analysis of ribosomes from rat liver, rabbit reticulocytes, and human HeLa cells.5,6,33 The identification of three exceptional ribosomal paralogues is described below. Ribosomal Protein L22-like 1 (L22L1) Was Identified as a Minor Ribosomal Component. In addition to the spots of known ribosomal proteins on the gel, a small spot near that of Journal of Proteome Research • Vol. 9, No. 3, 2010 1357

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Figure 3. Ribosomal protein L10L in testicular ribosomes. (A) Mass spectrum from the spot including ribosomal proteins L10 and L10L. Proteins from total cytoplasmic ribosomes from the mouse testis were separated by the modified RFHR method and visualized with colloidal Coomassie brilliant blue. The corresponding spot was subjected to MS after in-gel digestion with trypsin. Data of the numbered peaks were used for identification by Mascot search, resulting in identification of ribosomal protein L10L in addition to L10 (Table 4). The peaks c1, c5, and c9 are shown in a part of magnified spectrum in the inset. The m/z peaks in this spectrum correspond to singly charged peptides (MH+). (B) Amino acid sequence alignment of mouse L10 and L10L. The 100%-identical region between these two proteins is indicated by the open box. The peptide sequence corresponding to the L10L-specific peak b1 in (A) is indicated by the bar. The two L10L-specific peptide fragments, which can be obtained by complete tryptic digestion, are italicized. The lysyl residue that is specific for L10L and slowly cleaved by trypsin is indicated by the asterisk. The two residues of mouse L10, whose corresponding residues in human L10 are mutated in autism,63 are indicated by the arrowheads. Note that the peptide sequences of human and mouse L10 are completely identical.

ribosomal protein L22 (Entrez Gene ID, 19934) was detected from polysomes of the liver and mammary gland (Figure 2A). This satellite spot was not detected in polysomes from rat testis (data not shown). For comprehensive identification of ribosomal proteins, the satellite spots from the two different tissues were subjected to MS (Figure 2B and Table 2). PMF and MS/ 1358


MS indicated that both spots included a protein unpublished to date, ribosomal protein L22L1 (Entrez Gene ID, 68028). Ribosomal proteins L22 and L22L1 are encoded by distinct genes (Table 3), and the peptide sequence of L22L1 is 70% identical to that of L22 in mice (Figure 2C). Thus, it is conceivable that this minor paralogous protein, if detected on

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Figure 4. Analysis of the spots including ribosomal protein L39. The corresponding spots from ribosomes of the mouse liver (A) and testis (B) were subjected to MS after in-gel digestion with trypsin. The peaks specific for L39 (x1 and x2) are indicated by the arrowheads, and those specific for L39L (y1 and y2) are indicated by the arrows. The peaks x1 and x2 in (A) were used for identification by Mascot search, resulting in identification of ribosomal protein L39 (Supplementary Table 1). The peaks y1 and y2 in (B) were used for identification by Mascot search, resulting in identification of ribosomal protein L39L (Table 5). The m/z peaks in this figure correspond to singly charged peptides (MH+). (C) Amino acid sequence alignment of mouse L39 and L39L. The peptide fragments corresponding to the peaks x1, x2, y1, and y2 in (A) and (B) are indicated by the bars. The peptide fragment corresponding to the peak x2 contains a miscleaved tryptic site. The arginyl residues resistant to trypsin digestion are indicated by the asterisks.

electrophoresis gel, may not have been identifiable using conventional biochemical methods. Presence of Ribosomal Proteins L10L and L39L among Ribosomes Specifically from the Testis. In addition to its high sensitivity, another advantage of proteomic identification is that individual proteins can be found among protein mixtures. We also identified ribosomal proteins L10L (Entrez Gene ID, 238217) and L39L (Entrez Gene ID, 68172) among total cytoplasmic ribosomes of mouse testis (Figures 3A and 4B and Tables 4 and 5). L10L and L39L were found among spots including ribosomal proteins L10 (Entrez Gene ID, 110954) and L39 (Entrez Gene ID, 67248), respectively. L10L and L39L are paralogues of L10 and L39, respectively (Table 3). Despite the presence of these paralogues, no difference in the intensity or shape of the electrophoretic spot including L10 or L39 was observed between testis and the other two tissues (data not shown). These two pairs of homologues have quite high peptide

sequence similarity: 99% identical between L10 and L10L and 94% identical between L39 and L39L in mice (Figures 3B and 4C). Because of this similarity, these homologue pairs were inseparable even by the RFHR method. A peptide fragment corresponding to the peak b1 was a unique L10-specific peptide detected by MS (Figure 3A and Table 4). MS/MS confirmed its structure (Supplementary Figure 5). This fragment contained a miscleaved tryptic site. Complete tryptic digestion of L10L protein generated two peptides specific for this protein (residues 185-188 and 199-203, as italicized in Figure 3B), which were too small to be detected by PMF followed by MS/MS. The lysyl residue 184 is followed by the acidic aspartyl residue 185, where tryptic cleavage occurs slowly.47 This might have partly explained the incomplete digestion and successful detection of peak b1 by MS. Journal of Proteome Research • Vol. 9, No. 3, 2010 1359

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Table 4. Identification of Ribosomal Proteins L10 and L10L by MS (Figure 3A) identified peptide (PMF) peak

m/z obs.

a

m/z calc.

delta

missb

start-end

sequence

c1 c2 c3 c4 c5 c6 c7 c8 c9 c10

988.6304 3294.5203 3095.4080 1017.6558 993.5112 1187.7173 1251.7962 1267.7691 965.5914 1121.6928

988.5818 3294.5172 3095.3852 1017.5872 993.4372 1187.6411 1251.7122 1267.7071 965.5294 1121.6305

+0.0486 +0.0031 +0.0228 +0.0686 +0.0740 +0.0762 +0.0841 +0.0620 +0.0619 +0.0623

Common Peaks 1 31-38 1 41-69 0 43-69 0 91-98 0 102-110 0 117-128 0 129-139 0 129-139 0 146-153 1 146-154

a1 a2

1544.8406 1699.8463

1543.6501 1699.7512

+1.1906 +0.0952

L10-Specific Peaks 0 176-188 1 176-189

FNADEFEDMVAEK FNADEFEDMVAEKRc

b1

1482.7851

1482.6991

+0.0861

L10L-Specific Peak 1 176-188

FNADEFEDKVAAKc,d

IRIFDLGR AKVDEFPLCGHMVSDEYEQLSSEALEAAR VDEFPLCGHMVSDEYEQLSSEALEAAR LHPFHVIRc MLSCAGADR GAFGKPQGTVARc VHIGQVIMSIRc VHIGQVIMSIR (including M oxidation) EHVIEALR EHVIEALRR

a Each experimental m/z value (MH+) was transformed to a relative molecular mass (Mr) by the Mascot program. The Mr values are shown as “m/z obs.” in this table. b One missed cleavage was allowed in a Mascot search. c These peptide sequences were also identified by MS/MS analysis (Supplementary Table 1). d Two amino acid residues, which are different from the corresponding residues of L10, are underlined. MS/MS spectrum of this fragment is shown in Supplementary Figure 5.

Table 5. MS Data of Ribosomal Proteins L39 and L39L from the Testis (Figure 4B)a peptide (MS/MS) peak

x1

m/z obs.

b

1306.7781

y1 y2

1537.8848 1553.8726

m/z calc.

delta

miss

start-end

L39-Specific Peak 0 19-28

1306.7258

+0.0523

1537.8187 1553.8136

L39L-Specific Peaks +0.0661 0 19-30 +0.0590 0 19-30

sequence

QNRPIPQWIR QNRPIPQWIQMKc,d QNRPIPQWIQMKc (including M oxidation)

a Ribosomal protein L39 had been identified by MS/MS from the liver (Figure 4A and Supplementary Table 1). Ribosomal protein L39L was identified by the present data. b Each experimental m/z value (MH+) was transformed to a relative molecular mass (Mr) by the Mascot program. The Mr values are shown as “m/z obs.” in this table. c One amino acid residue, which is different from the corresponding residue of L39, is underlined. d MS/MS spectrum of this fragment is shown in Supplementary Figure 6.

L39 and L39L are very small basic proteins (Table 3). The peptide fragments corresponding to peak x1 of L39 and peak y1 of L39L (Figure 4A,B) are unique, and have a mass large enough for detection by MS after complete tryptic digestion. The MS/MS spectrum of the L39L-specific peak y1 is shown in Supplementary Figure 6. It should be noted that the arginyl residues within these peptides (indicated by the asterisks in Figure 4C) are followed by proline, and resistant to cleavage by trypsin.47 Neither L10L nor L39L was detected from polysomes of the liver or mammary gland (Figure 4A and data not shown). RT-PCR analysis of normal mouse tissues showed predominant expression of ribosomal proteins L10L and L39L in the testis at the message level (Figure 5). Public DNA microarray databases support their restricted expression (L10L, http://biogps.gnf.org/ #goto)genereport&id)238217; L39L, http://biogps.gnf.org/ #goto)genereport&id)68172). Collectively, it appears that L10L and L39L are ribosomal components unique to the testis. Mouse L39L Is Included among Translating Ribosomes and Is an Apparent Counterpart of Human L39L. A human protein highly homologous to ribosomal protein L39 has been reported,12,21 and is listed in the Entrez Gene database as human ribosomal protein L39L (Entrez Gene ID, 116832). The human and mouse L39L proteins are 90% identical, which is lower than the homology between the human and mouse L39 1360


proteins (100%) (Figure 6A). A common substitution in the two L39L proteins is present at one residue, R28Q. Even though no other paralogues of L39 have been identified in mice, this feature led to the suspicion that human L39L and mouse L39L might have evolved independently to play different roles including extraribosomal functions.5,48 Because the precise role of L39L in humans has been vague, it has been impossible to judge precisely whether human L39L and mouse L39L are orthologous. Nonetheless, since some properties of the human protein related to its functions have been described, the latter possibility might be evaluable based on these data. For example, RT-PCR has shown that human L39L is expressed predominantly in the testis.12,21 Mouse L39L shares the same tissue distribution, as described above. In human cultured cells transiently transfected with a vector expressing human L39L, (i) human L39L was localized mainly in the nucleolus, where ribosome biogenesis occurs, and (ii) this protein was included in the large subunit of the ribosome in the cytoplasm.21 We therefore tested whether mouse L39L possessed these two properties. To clarify the intracellular localization of mouse L39L, cDNA of this protein was subcloned into a vector to facilitate expression of the protein tagged with GFP at its N- or C-terminus in mammalian cells, and transiently transfected into human 293T cells. Irrespective of the position of the GFP

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tag, L39L was observed in the nucleus (panels in the left low of Figure 7 and data not shown). Very strong signals were detected from nucleoli. The nucleolar localization of L39L was confirmed by colocalization with nucleolar fibrillarin. No specific localization in the nucleolus was observed in cells expressing GFP (panels in the right low of Figure 7). Intracellular localization was also examined by transfection of a vector expressing L39L fused with a much smaller peptide, a FLAG tag, in the same manner. Although, for some reason, cells expressing FLAG-tagged L39L were reproducibly much fewer than those expressing GFP-tagged L39L, the main positive signals were observed in the nucleoli of the transfected cells (data not shown). Newly synthesized ribosomal proteins are reportedly transferred to the nucleolus,49,50 and marked accumulation of a ribosomal protein in the nucleolus has been shown to occur under transient expression conditions.21,51 The intracellular localization of mouse L39L was very similar to that of human L39L and other ribosomal proteins. After transfer to the nucleolus, ribosomal proteins are incorporated into the precursors of ribosomal subunits, exported through the nuclear pores, and involved in translation as a component of the cytoplasmic ribosome.49,52,53 The nucleolar localization of mouse L39L prompted us to examine whether this protein is incorporated in translating ribosomes, that is, polysomes, in the testis. To obtain an antibody binding specifically to mouse L39L, a peptide unique to this protein (indicated in Figure 6A) was used to immunize four female mice. The antiserum obtained was reactive with recombinant mouse L39L protein, but not with L39 protein (Figure 6B), and showed positive reactivity with the mouse testis ribosome including L39L, but not with the liver ribosome (Figure 6C). The protein reactive with this antiserum migrated more slowly than calculated on the gel (Table 3). Although this irregular mobility cannot currently be explained, many highly basic proteins (isoelectric point >11) have been reported to migrate more slowly than expected.54 Immunoblotting of whole lysates of mouse testis with this antiserum yielded a putative positive band within the background staining (data not shown), probably as a result of nonspecific binding of the antibody. Because the mouse L39L antigenic peptide differs by three (out of 13) residues from the corresponding peptide of human L39L (Figure 6A), this antiserum seems to exhibit poor reactivity with human L39L. These findings indicate that the antiserum reacts specifically with L39L upon immunoblotting of purified mouse ribosomes. Total cytoplasmic ribosomes were purified from mouse testis and subjected to polysomal profiling using sucrose gradient ultracentrifugation. A protein reactive with anti-L39L antiserum was detected in the fractions including the 60S large subunit, the 80S monosome, and polysomes, as shown for ribosomal protein L10 (Figure 8A). Inclusion of L39L in the large subunit was confirmed by profiling after subunit dissociation with puromycin (Figure 8B). This profiling analysis indicates the involvement of mouse L39L in translation as a component of the ribosomal large subunit and supports the orthologous relationship between human L39L and mouse L39L. Figure 5. Specific expression of ribosomal proteins L10L and L39L in the testis. (A) Expression of ribosomal proteins L10L and L10 in mouse tissues was analyzed by RT-PCR. (B) Expression of ribosomal proteins L39L, L39, S5, and L12 in mouse tissues was analyzed by quantitative RT-PCR. The expression levels are presented on a linear scale and in arbitrary units. The representative results of two independent experiments are shown. The detailed procedures for RT-PCR including quantitative analysis are described in the Methods section.

Discussion In this study, we performed proteomic analysis of ribosomes in three different rodent tissues and found that ribosomal proteins L10L, L22Ll, and L39L are components of ribosomes. The ribosome is composed of the small and large subunits, which are conserved in the three domains of living organisms: eubacteria, archaebacteria, and eukaryotes. The three identified Journal of Proteome Research • Vol. 9, No. 3, 2010 1361

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Figure 6. Design and evaluation of a probe for mouse ribosomal protein L39L. (A) Comparison of the peptide sequences of ribosomal proteins L39 and L39L in humans and mice. Sequence homologies between the indicated pairs are shown on the right. The antigenic peptide sequence of anti-mouse L39L antibody is indicated by the bar. (B) Specific reactivity of anti-mouse L39L antiserum against recombinant proteins. Mouse L39L fused with thioredoxin, human/mouse L39 fused with GST, and thioredoxin were produced in bacteria and subjected to SDS-PAGE followed by immunodetection with each antibody against the protein indicated on the right. Trx, thioredoxin. (C) Reactivity of anti-mouse L39L antiserum against ribosomal proteins from the testis and liver. Cytoplasmic ribosomes purified from the testis and liver of mice were subjected to SDS-PAGE with 20% gel and high-molarity buffers and immunodetection with anti-L39L antiserum and antibody against ribosomal protein L10. Migration positions of molecular mass markers are indicated on the left.

proteins are paralogues of proteins in the 60S large ribosomal subunit. This is indicative of the greater evolutionary stability of proteins in the small subunit on the domain scale.55 However, our findings do not indicate that paralogues of a ribosomal protein in the 40S small subunit are absent in other cellular contexts. In fact, ribosomal protein S27-like, a paralogue of ribosomal protein S27,56 is reportedly expressed at the protein level, although its inclusion in the ribosome has not been examined.57 We tried to detect this paralogous protein around the spots including S27 on electrophoresis gel, but were unsuccessful (Supplementary Figure 9). This may have been attributable to the fact that the gene encoding S27-like is directly activated by p53,57 a tumor suppressor that is maintained at a low level by proteasomal degradation in normal viable cells.58 Ribosomal protein L22L1 was detected as an isolated spot near L22 (Figure 2A). The calculated molecular mass and isoelectric point of these two proteins are listed in Table 3. The relatively large differences between them are likely to contribute to the separation of L22 and L22L1 on gel. L22L1 was detected in mammary gland and liver tissue. This seems consistent with the broad expression of mRNA for this protein evident in public DNA microarray databases (http://biogps.gnf.org/#goto) genereport&id)68028). L22 and L22L1 belong to the ribosomal 1362


protein L22e family, one of the families that are conserved between archaebacteria and eukaryotes, and are not present in eubacteria. Orthologous L22 has been reported to be dispensable for translation in vitro,59 and null mutants for this protein are viable.60 Thus, the precise function of the L22e family in ribosomes remains unclear. The activity of ribosomes in protein synthesis, either in the absence of L22Ll or after its overexpression, needs to be tested. In contrast to identification of L22L1 from two different tissues, L10L and L39L were indicated to be expressed specifically in the testis. Their specific expression at the message level (Figure 5) has been mentioned in a previous report,12 which, in addition to L10L and L39L, suggested that mRNA for the ribosomal protein L36a-like, a paralogue of ribosomal protein L36a, was expressed in mice. Unfortunately, because the peptide sequences of L36a-like and L36a are 100% identical in mice (99% identical in human), we were unable to discriminate these translated products. Ribosomal protein L10 (also called Qsr1p) was originally discovered in yeast and is required for nuclear export of the large subunit and for ribosomal subunit joining.52,61 It has been proposed that L10 belongs to the L10e family, shared by ribosomes from the three evolutionary domains.55,62 The eubacterial and archaeal counterparts of L10, L16 and L10e,


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Figure 7. Localization of newly synthesized mouse ribosomal protein L39L in the nucleolus. Human 293T cells were transiently transfected with the vector for the expression of N-terminally GFP-tagged L39L and a mock vector expressing GFP and cultured for 24 h. After fixation, permeabilization, and treatment with antibody against fibrillarin, a nucleolar maker, cells were observed under a confocal laser microscope. Bar, 10 µm. DIC, differential interference contrast.

respectively, have extensions that reach toward the peptidyltransferase center of the large subunit,3 and a similar location of L10 was observed in the yeast ribosome.4 Therefore, these family members may play a fundamental role in translation. Although the difference in peptide sequence between mouse L10 and L10L is restricted to the C-terminal region (Figure 3B), deletion or mutations of this region affect ribosome function in yeast.61,62 Two point mutations in this region of human L10 (L206 M and H213Q; the corresponding residues in mouse L10 are indicated by the arrowheads in Figure 3B) are connected to a tissue (hippocampal brain)-specific phenotype, autism.63 The present analysis of L10L might help to shed further light on this multifunctional family. Ribosomal protein L39L was found to be included in translating ribosomes in the testis (Figure 8A). This raises the question of how many ribosomes including L39L exist in this tissue. The mRNA levels of L39 and L39L in tissues can be roughly estimated from our quantitative RT-PCR data. The RTPCR conditions for L39 and L39L were quite similar; the

sequences of the PCR primers were identical except for a few nucleotides (Table 1), and the amplified cDNAs shared 83% sequence identity (Supplementary Figure 8). Given the same amplification efficiency under these two conditions, the amounts of these products can be estimated from the Ct values in quantitative PCR (Table 6). This rough evaluation suggests that the mRNA levels of these two proteins are comparable in the testis. This comparable expression is unlikely due to transcriptional repression of L39 in the testis, because the expression patterns of L39 among tissues are similar to those of other ribosomal proteins (Figure 5B). Coexpression of L39 and L39L mRNAs has also been suggested in the human testis.12,21 The mass spectra of the spots including L39 and L39L showed that the intensity of the peak unique to L39 was comparable to that of the corresponding peak of L39L (peaks x1 and y1, respectively, in Figure 4B; another experiment is shown in Supplementary Figure 7). Taken together, the data suggest that L39L is not a minor ribosomal component in the testis. Journal of Proteome Research • Vol. 9, No. 3, 2010 1363

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Sugihara et al. the majority of ribosomal proteins, L39L is incorporated into a nucleolar ribosomal precursor. Hence, it can be speculated that, in the testis, L39 and L39L may be expressed within the same cell to compete for inclusion in a preribosomal complex in the nucleolus, resulting in the presence of L39L instead of L39 in some of the large subunits. Another possibility is that these two proteins are expressed in distinct cells within the tissue. Although questions still remain to be answered, the present study has revealed the presence of hitherto unknown heterogeneity in the ribosome.

Figure 8. Mouse ribosomal protein L39L is included in the large subunit of the translating ribosome. (A) Total cytoplasmic ribosomes from the mouse testis were ultracentrifuged at 37 000 rpm for 3.0 h at 4 °C on 15-45% sucrose density gradients. (B) After subunit dissociation with puromycin, total testicular ribosomes were ultracentrifuged at 38 000 rpm for 4.0 h at 25 °C on 10-40% sucrose density gradients. RNA was detected by absorbance at 254 nm. Fractions were immunoblotted using antibodies indicated on the left. Ribosomal proteins L10 and S6 are markers of the 60S large subunit and the 40S small subunit, respectively. Table 6. Coexpression of Ribosomal Proteins L39 and L39L in the Testis experiment no.

1c 2

Cta tissue

L39

L39L

L39L/L39 mRNA expression ratiob

liver testis liver testis

19.8 20.5 17.3 20.8

36.8 18.8 32.8 18.2

7 × 10-6 3.2 2 × 10-5 6.1

a Ct values were obtained in two independent experiments of quantitative RT-PCR. b Calculated by 2-(CtL39L-CtL39). See text for assumptions in this rough estimation. c All the results in experiment no. 1 are shown in Figure 5B.

Apart from ribosomal proteins P1 and P2, which form an oligomeric complex, ribosomal proteins are included in the eukaryotic ribosome in equimolar amounts, that is, one protein copy per ribosome.5 L39 and L39L have high structural similarity (Figure 6A). Nucleolar accumulation of L39L (Figure 7) is same as that of human/mouse L39,21 thus suggesting that, like 1364


The question then arises as to whether the structural heterogeneity of testicular ribosomes is connected to translation or testis biology. Functional paralogues in mammalian ribosomes reported to date have been very rare, but an example is human ribosomal proteins S4X and S4Y. The former gene is located on the X chromosome, and the latter on the Y chromosome. The S4 gene on the Y chromosome has not been found in the mouse genome. The peptide sequence of S4X is 93% identical to that of S4Y. These proteins have been studied intensively in relation to Turner syndrome.64 S4X and S4Y are expressed ubiquitously, and S4Y has been reported to be 15% as abundant as S4X in ribosomes from normal human cells, indicating structural heterogeneity in male ribosomes.65 However, S4X and S4Y have been shown to have identical translational function,66 which does not support a special role of S4Y in male ribosomes. While the gene of ribosomal protein L39L has been found only in mammals, that of L39 is conserved between archaebacteria and eukaryotes.5,55 Because no eubacterial counterpart of L39 exists, L39 is considered to play a regulatory role in ribosomes.20 Although ribosomal protein L39 is actually nonessential in budding yeast, the ribosome lacking L39 shows low translational accuracy.67 Structural studies of the ribosome exit tunnel have suggested that L39 is included in a tunnel sensor recognizing the side-chains of nascent polypeptides.68,69 The genes of L39L, and also L10L, are considered to have been generated by retrotransposition of their original genes (L39 and L10, respectively) located on the X chromosome.12 Traffic of many other genes from the X chromosome to autosomes has been found in the human and mouse genomes.70 One of these retrogenes, Utp14b, whose yeast homologue is involved in ribosome biogenesis, has been demonstrated to be required for spermatogenesis in mice.71 It is therefore conceivable that ribosomes including L39L function in testis-specific phenomena such as spermatogenesis through modulation of translation. Mouse testicular ribosomes, including L10L and L39L, seem to provide a good model for understanding the relationship between ribosomal heterogeneity and translation, and for examining the importance of such heterogeneity in vivo. In conclusion, the structural heterogeneity that has been demonstrated in this study is far less extensive than that in plant and yeast ribosomes, consistent with published biochemical and genetic data for mammalian ribosomes. Nevertheless, the heterogeneity of testicular ribosomes does not seem to be negligible. In the present study, we focused on heterogeneity in ribosomes from normal tissues. When considered in the light of recent data pertaining to the relationship between the ribosome and diseases,19–21,48,58,63 it is anticipated that proteomic studies of ribosomes under pathological conditions will reveal limited but significant heterogeneity. Abbreviations: Ct, threshold cycle; GFP, green fluorescent protein; GST, glutathione S-transferase; L10L, L10-like; L22L1, L22-like 1; L39L, L39-like; MS, mass spectrometry; MS/MS,

Ribosomal Heterogeneity in Mammals tandem mass spectrometry; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; PMF, peptide mass fingerprinting; RFHR, radical-free and highly reducing; RT, reverse transcription; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Acknowledgment. We thank Yosuke Kishino, Mariko Kugo, and Yuki Taga (Nagoya University) for helpful supports. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by research grants from the Nakatomi Foundation, the Mishima Kaiun Memorial Foundation, and the Life Science Foundation of Japan. Supporting Information Available: Supplementary Figure 1, polysomal profile of total cytoplasmic ribosomes from the mouse liver. Supplementary Figure 2, ribosomal proteins separated by the standard RFHR method. Supplementary Figure 3, magnified views of ribosomal protein S6 on gel. Supplementary Figure 4, improved detection of very small proteins after electrophoresis at low temperature. Supplementary Figure 5, MS/MS spectrum of the peak b1 in Figure 3A. Supplementary Figure 6, MS/MS spectrum of the peak y1 in Figure 4B. Supplementary Figure 7, mass spectrum from a spot including ribosomal proteins L39 and L39L in another experiment. Supplementary Figure 8, comparison of the cDNA sequences of mouse ribosomal proteins L39 and L39L. Supplementary Figure 9, amino acid sequence alignment of mouse ribosomal proteins S27 and S27-like. Supplementary Table 1, mouse ribosomal proteins identified by MS in this study. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Palade, G. E. A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol. 1955, 1, 59–68. (2) Noller, H. F. Structure of the bacterial ribosome and some implications for translational regulation. In Translational Control in Biology and Medicine; Mathews, M. B., Sonenberg, N., Hershey, J. W. B., Eds.; Cold Spring Harbor Laboratory Press: New York, 2007; pp 41-58. (3) Steitz, T. A. A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Biol. 2008, 9, 242–253. (4) Taylor, D. J.; Frank, J.; Kinzy, T. G. Structure and function of the eukaryotic ribosome and elongation factors. In Translational Control in Biology and Medicine; Mathews, M. B., Sonenberg, N., Hershey, J. W. B., Eds.; Cold Spring Harbor Laboratory Press: New York, 2007; pp 59-85. (5) Wool, I. G.; Chan, Y.-L.; Gluck, A. Mammalian ribosomes: the structure and the evolution of the proteins. In Translational Control; Hershey, J. W. B., Mathews, M. B., Sonenberg, N., Eds.; Cold Spring Harbor Laboratory Press: New York, 1996; pp 685732. (6) McConkey, E. H.; Bielka, H.; Gordon, J.; Lastick, S. M.; Lin, A.; Ogata, K.; Reboud, J. P.; Traugh, J. A.; Traut, R. R.; Warner, J. R.; Welfle, H.; Wool, I. G. Proposed uniform nomenclature for mammalian ribosomal proteins. Mol. Gen. Genet. 1979, 169, 1–6. (7) Woolford, J. L., Jr.; Warner, J. R. The ribosome and its synthesis. In The Molecular and Cellular Biology of the Yeast Saccharomyces; Broach, J. R., Pringle, J. R., Jones, E. W., Eds.; Vol. 1; Cold Spring Harbor Laboratory Press: New York, 1991; pp 587-626. (8) Planta, R. J.; Mager, W. H. The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast 1998, 14, 471–477. (9) Uechi, T.; Tanaka, T.; Kenmochi, N. A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders. Genomics 2001, 72, 223–230. (10) Balasubramanian, S.; Zheng, D.; Liu, Y. J.; Fang, G.; Frankish, A.; Carriero, N.; Robilotto, R.; Cayting, P.; Gerstein, M. Comparative analysis of processed ribosomal protein pseudogenes in four mammalian genomes. Genome Biol. 2009, 10, R2.

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