Identification of the methylated ribosomal proteins in HeLa cells and the fluctuation of methylation during the cell cycle

Chang, F. N.; Navickas, I.J.; Au, Clement; Budzilowicz, Carol

doi:10.1016/0005-2787(78)90118-1

Cited by 25 publications

(19 citation statements)

References 16 publications

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“…In addition to heterogeneity derived from different ribosomal genes for the same subunit, covalent modification introduces further potential differences in ribosomal protein structure and function. Covalent modifications of ribosomal proteins have been reported in bacteria (28,29,(31)(32)(33)(34)(35), fungi (3, 4, 8, 36 -38), mammals (5)(6)(7)39), and plants (19). However, in almost all of these studies, limitations of the techniques used, such as peptide mass fingerprinting, topdown MS analysis of intact r-proteins, and acid hydrolysis to remove rRNA, have precluded detection of acid/base-labile, low stoichiometry, or difficult-to-detect modifications or the determination of precise details about the exact position(s) and structure(s) of the modified residue(s).…”

Section: Covalent Modifications Of Arabidopsis 80 S Ribosomal Proteinmentioning

confidence: 99%

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Carroll¹,

Heazlewood²,

Ito³

et al. 2008

Molecular & Cellular Proteomics

184

253

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Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical genespecific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene-or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co-and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These sitespecific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis. Molecular & Cellular Proteomics 7:347-369, 2008.Ribosomes are large ribonucleoprotein complexes that catalyze the peptidyltransferase reaction in polypeptide synthesis and are thus responsible for the translation of transcripts encoded in cellular genomes. These complexes play the most fundamental role of any protein complex in the generation of the cellular proteome as a whole. Ribosomes consist of two subunits, large and small, but the internal composition of these subunits and their macromolecular size varies between bacteria, animals, fungi, and plants. Both these subunits are composed on rRNA and protein (r-protein) 1 components. Among eukaryotes the 80 S cytosolic ribosomes of the yeast (Saccharomyces cerevisiae), rat (Rattus norvegicus), and human (Homo sapiens) have been the most extensively investigated. These studies have revealed four distinct rRNAs, the 18 S rRNA of the 40 S subunit and 5, 5.8, and 23 S rRNAs of the 60 S subunit. Up to 79 distinct proteins are part ...

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Section: Covalent Modifications Of Arabidopsis 80 S Ribosomal Proteinmentioning

confidence: 99%

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Carroll¹,

Heazlewood²,

Ito³

et al. 2008

Molecular & Cellular Proteomics

184

253

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“…The Type I enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric N G ,NЈ G -dimethylarginine residues. In mammals, there are five Type I enzymes (PRMT1,3,4, 6 and 8).Proteins that are substrates for the PRMTs usually contain glycine and arginine-rich patches, GAR motifs, that are the sites of methylation. The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins. Thirty years ago, HeLa cell ribosomal proteins were shown to be heavily lysine-and arginine-methylated (2), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3). PRMT3 is a ribosome-associated protein (4,5) and may be responsible for much of the arginine methylation that occurs in this molecular machine.…”

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confidence: 99%

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Ribosomal Protein rpS2 Is Hypomethylated in PRMT3-deficient Mice

Swiercz

Cheng

Kim

et al. 2007

Journal of Biological Chemistry

121

100

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PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.Arginine methylation is a common posttranslational modification that occurs in both the nucleus and the cytoplasm (1). The methylation of arginine residues is catalyzed by at least two different classes of protein arginine methyltransferase (PRMT) 2 enzymes. The Type I enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric N G ,NЈ G -dimethylarginine residues. In mammals, there are five Type I enzymes (PRMT1,3,4, 6 and 8).Proteins that are substrates for the PRMTs usually contain glycine and arginine-rich patches, GAR motifs, that are the sites of methylation. The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins. Thirty years ago, HeLa cell ribosomal proteins were shown to be heavily lysine-and arginine-methylated (2), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3). PRMT3 is a ribosome-associated protein (4, 5) and may be responsible for much of the arginine methylation that occurs in this molecular machine.PRMT3 was identified as a PRMT1 binding protein in a yeast two-hybrid screen (6). However, gel filtration analysis of RAT1 cells demonstrated that PRMT3 occurs as a monomer and is not complexed in vivo with PRMT1 (6). PRMT3 also interacts with the tumor suppressor DAL-1 (7). This interaction inhibits PRMT3 enzymatic activity, both in in vitro reactions and in cell lines. At the organ level, PRMT3 is ubiquitously expressed (6), but in the brain it displays higher expression in neurons than in glial cells (8). PRMT3 is the only type 1 arginine methyltransferase that is localized exclusively to the cytoplasm (6, 9). Another unique property of PRMT3 is that it harbors a zinc finger domain at its N terminus. It has been proposed that this domain may play a role in the regulation of PRMT3 activity or in the recognition of PRMT3 substrates (6, 10). Deletion analysis studies demonstrated that PRMT3 lacking the zinc finger domain is still active in vitro, however the zinc finger minus enzyme loses its abil...

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“…At least seven 40s proteins from HeLa cells were found to be methylated, and the level of their methylation was shown to be altered in the Abbreviations. MALDI, matrix-assisted laser-desorption ionization ; Lys-C, endoproteinase Lys-C; RP, reverse-phase; rp, ribosomal protein ; S-proteins, proteins of the small ribosomal subunit; 40S, small ribosomal subunit; TP40, total protein mixture of the small ribosomal subunit; TOF, time-of-flight ; Pth-Xaa, phenylthiohydantoin -amino-acid.course of the cell cycle [12]. It is also known that ribosomal proteins may be acetylated 17, 13, 141.…”

mentioning

confidence: 99%

Characterization of the Human Small‐Ribosomal‐Subunit Proteins by N‐Terminal and Internal Sequencing, and Mass Spectrometry

Vladimirov

Ivanov

Карпова

et al. 1996

European Journal of Biochemistry

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Reverse-phase HPLC was used to fractionate 40s ribosomal proteins from human placenta. Application of a C, reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40s human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40s proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.Keywords: human ribosomal proteins ; HPLC ; N-terminal and internal amino acid sequencing ; matrixassisted-laser-desorption ionization MS ; post-translational modifications.Human ribosornd proteins are the main components of the fundamental processes which translate genetic information into proteins at the ribosome. Some ribosomal proteins (rp) are further involved in non-ribosomal functions. Recently, rp L7 was shown to contain the basic-region-leucine-zipper sequence which mediates the stable binding of this protein to DNA [I]. Moreover, rp S3 was found to act as an endonuclease in the DNA-repairing system [2]. Finally, some data indicate that tumor diseases may be accompanied by overexpression of rp genes 13, 4) and by chromosomal rearrangements in regions of rp genes [ S ] . Therefore, structural analysis of these proteins is necessary for complete understanding of their fundamental role in the cell. The task of structure determination of rat rp has been almost fully resolved by the application of recombinant-DNA technology ; details are reviewed in [6]. However, knowledge of the cDNA sequences does not provide information concerning post-translational modifications of the proteins after their biosynthesis and assembly into the ribosomal subunits. Several modifications of rp have been determined in the eubacterial ribosome [7, 81, and the eukaryotic rprotein S6 is known to be phosphorylated 191. This phosphorylation was shown to be connected with cell growth [I 01 and cell cycle control [I I]. At least seven 40s proteins from HeLa cells were found to be methylated, and the level of their methylation was shown to be altered in the Abbreviations. MALDI, matrix-assisted laser-desorption ionization ; Lys-C, endoproteinase Lys-C; RP, reverse-phase; rp, ribosomal protein ; S-proteins, proteins of the small ribosomal subunit; 40S, small ribosomal subunit; TP40, total protein mixture of the small ribosomal subunit; TOF, time-of-flight ; Pth-Xaa, phenylthiohydantoin -amino-acid.course of the cell cycle [12]. It is also known that ribosomal proteins may be acetylated 17, 13, 141. However...

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Identification of the methylated ribosomal proteins in HeLa cells and the fluctuation of methylation during the cell cycle

Cited by 25 publications

References 16 publications

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Ribosomal Protein rpS2 Is Hypomethylated in PRMT3-deficient Mice

Characterization of the Human Small‐Ribosomal‐Subunit Proteins by N‐Terminal and Internal Sequencing, and Mass Spectrometry

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