Characterization and Analysis of Posttranslational Modifications of the Human Large Cytoplasmic Ribosomal Subunit Proteins by Mass Spectrometry and Edman Sequencing

Schizosaccharomyces pombe Rmt3 is a member of the proteinarginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show misregulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.Protein arginine methylation is a posttranslational modification catalyzed by a family of enzymes known as protein-arginine methyltransferases (PRMTs).2 Although protein-arginine methyltransferase activity has never been demonstrated in prokaryotic organisms, genes encoding PRMTs have been identified in a variety of unicellular and multicellular eukaryotes (1, 2). In humans, 10 PRMTs have so far been identified (3). Most PRMTs are divided in two major classes, depending of the type of dimethylarginine they produce. Whereas both type I and II PRMTs use S-adenosyl-L-methionine as a cofactor for the monomethylation of specific arginines within substrate proteins, type I and type II enzymes can also produce asymmetricarginine, respectively (1). Interestingly, protein arginine methylation is often found within arginine-glycine (RG)-rich regions of nucleic acid-binding proteins (4). The functional role of PRMTs is likely to be mediated by the modification of substrate proteins. Accordingly, proteins involved in specific steps of gene expression, including transcription (5, 6), splicing (7), polyadenylation (8, 9), mRNA export (10), and translation (11-13), are modified by arginine methylation. Methylation of specific arginine residues within the N-terminal tails of nucleosomal histones is also important for gene regulation and chromatin remodeling (14, 15), thereby influencing biological processes, such as cell fate determination (16) and oncogenesis (17). As yet, however, the biological role of most PRMTs remains poorly understood.The ribosome is the macromolecular complex responsible for protein synthesis in all living cells. In eukaryotes, the 80 S ribos...

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confidence: 72%

A Methyltransferase-independent Function for Rmt3 in Ribosomal Subunit Homeostasis

Perreault

Gascon

D'Amours

et al. 2009

“…and Rpl42ab proteins appear to be modified (41). The only human large subunit ribosomal protein known to be modified by lysine methylation is L29 (41); the yeast Rpl29 ortholog is unmodified (18).…”

Section: Discussionmentioning

confidence: 99%

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

Webb¹,

Al-Hadid²,

Zurita-Lopez

et al. 2011

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using topdown mass spectrometry. Lysine modification was confirmed by detecting 3 H-N-⑀-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-L-[methyl-3 H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-␤-strand methyltransferase, results in the loss of the ؉14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-⑀-monomethyllysine formation within Rpl1ab on ribosomes from the ⌬YLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38 -53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.Proteins of the eukaryotic translational apparatus are often targets for post-translational covalent modifications (1). One of the major types of these reactions is the transfer of methyl groups from S-adenosylmethionine to a variety of residues including arginine (2-5), lysine (6 -11), glutamine (12), histidine (13), and N-terminal (14, 15) residues. Ribosomal proteins (1-5, 7-11, 13, 14, 16), elongation factor 1A (1, 6, 17), and translational release factors (12) are common substrates of protein methyltransferases. These modifications are known to enhance resistance to ribosome-targeting antibiotics, facilitate ribosomal protein transport into and out of the nucleus, allow efficient ribosomal assembly, and increase translational accuracy (1, 5).We have been interested in understanding the role of protein methylation in ribosomal function and assembly in Saccharomyces cerevisiae using a proteomics-guided approach. Previous efforts have identified modifications within proteins of the large ribosomal subunit. Interestingly, mass spectrometric analysis suggested that six proteins, Rpl1ab, Rpl3, Rpl12ab, Rpl23ab, Rpl42ab, and Rpl43ab, 3 were subject to methylation (18). Further analysis has localized the methylation sites on four of these proteins. Rpl3 is modified to form a 3-methylhistidine residue at position 243 (1...

“…A recent study further demonstrated that the monomethylation at Lys-40 and Lys-55 in Rpl42 is dependent on two other SET proteins, the Ybr030w gene product and Set7, respectively (16), although the direct enzymatic activity of these proteins has yet to be demonstrated. Several mass spectrometric studies have also identified methyl modifications on ribosomal proteins in plants and mammals (17)(18)(19). Although ribosomal protein methylation appears to be conserved among different organisms, the physiological roles of these lysine methylations remain to be fully elucidated.…”

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confidence: 99%

Methylation of Ribosomal Protein L42 Regulates Ribosomal Function and Stress-adapted Cell Growth

Shirai

Sadaie

Shinmyozu³

et al. 2010

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of nonhistone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in ⌬set13 mutant cells. ⌬set13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.Post-translational modifications regulate protein structure and function, and one of the most common is lysine methylation (1). Although lysine methylation does not change the overall charge of the side chain, it can influence the activity of protein by providing a novel interface for its interactions with other molecules. Most protein lysine methylations, with a few exceptions, are catalyzed by the SET domain-containing protein (SET protein) family (2). The SET domain was originally identified in three Drosophila proteins, Su(var)3-9, Enhancer of zest, and Trithorax, and was later demonstrated to be the responsible domain for histone-lysine methyltransferase function (3). The characterization of these enzymes has revealed the roles of histone lysine methylation in many different biological processes, including higher order chromatin assembly and transcriptional regulation (4). Recent studies have shown that histone lysine methylation is directly reversed by several histone demethylase families (5).Lysine methylation has also been identified in non-histone proteins that include ribulose 1,5-bisphosphate carboxylase/ oxygenase in plants (6), cytochrome c in yeast (7), mammalian TAF10 and p53 (8 -10), and ribosomal proteins in a diverse range of species (11). The methylation of ribosomal proteins has been observed in both prokaryotes and eukaryotes. In the budding yeast Saccharomyces cerevisiae, a combination of in vivo labeling and direct mass spectrometric analysis of the ribosomal proteins revealed that six of them, Rpl1, Rpl3, Rpl12, Rpl23, Rpl42, and Rpl43, are post-translationally methylated (12, 13). By analyzing the methylation state of S. cerevisiae mutant strains with deletions in candidate SET domain-containing genes, tw...