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

Abstract

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...

Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae : Roles in ribosome biogenesis and translation

“…2). Increased sensitivity of rkm4 Δ strains to cycloheximide has previously been reported [20,21]. No differences in sensitivity of the mutants were seen on paramomycin-containing agar plates, compared to wild type (data not shown).…”

Ribosome Collision Is Critical for Quality Control during No-Go Decay

“…Motivated by these observations, we sought to explore whether ribosome monoubiquitination is also triggered by ribosome collision. To accomplish this, we took advantage of the fact that substituting proline for glutamine at residue 56 (P56Q) of ribosomal protein RPL42 (eL42) renders ribosomes resistant to cycloheximide (Roguev et al, 2007; Shirai et al, 2010). Fortuitously, in yeast RPL42 is encoded by two paralogues, RPL42A and RPL42B , and as a consequence mutating one should result in a mixed population of ribosomes with respect to cycloheximide sensitivity.…”

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

“…When grown in a competition with a parent strain in minimal medium, the YLR137W knock-out shows a slight growth advantage (relative fitness score is 1.003) (46). In both S. cerevisiae and S. pombe, cells lacking methylation at lysine 55 of Rpl42 are more sensitive to cycloheximide (9,11), and S. pombe mutants have survival defects in stationary phase (11). The growth defect in S. pombe resulting from the overexpression of the enzyme responsible for lysine 3 methylation of Rpl12, as well as the nucleolar localization of the enzyme, suggests a role for methylation in ribosomal assembly.…”

“…In Schizosaccharomyces pombe, enzyme orthologs of S. cerevisiae Rkm2 (10) and Rkm4 (11) catalyze the methylation of lysine 3 of Rpl12 and lysine 55 of Rpl42, respectively. In the higher plant A. thaliana, lysine 3 of L12 is trimethylated, and lysine 55 of L36a (the Rpl42 ortholog) is monomethylated (8) as seen in S. cerevisiae (9) and S. pombe (10).…”