Identification and Biological Characterization of Ribosomal Protein Methyltransferases in Yeast and Humans

Post-translational modifications (PTMs) of proteins is key to the functionality of complex cellular processes. Methylation is one of the most common PTMs in nature. I have focused my work on protein methylation reactions that affect the translational machinery decoding messenger RNA sequences to protein sequences in the yeast Saccharomyces cerevisiae. The biological roles of methylation of the messenger, transfer, and ribosomal RNA components of the translational machinery have been extensively studied and characterized. However, much less is known about the prevalence and the biological roles of ribosomal protein methylation and the methyltransferases responsible for these modifications. In this dissertation, I aimed to identify and characterize ribosomal protein methyltransferases in S. cerevisiae and humans and uncover their biological roles. Using intact mass spectrometry of ribosomal proteins isolated from wild type and putative methyltransferase mutants, the enzymes responsible for methylating the ribosomal proteins were identified. The methylation sites on the ribosomal proteins were then pinpointed using top-down and/or bottom-up mass spectrometry followed by amino acid analysis of radiolabeled ribosomal proteins using cation-exchange and thin-layer chromatography. We show that the yeast enzyme Ntm1 (N-terminal methyltransferase 1) is responsible for methylating ribosomal proteins Rpl12ab and Rps25a/Rps25b at their N-terminus. BLAST analyses identified homologs of Ntm1 in higher eukaryotes, including humans (METTL11A). Methylation assays using yeast and mammalian extracts or recombinant forms of Ntm1 and METTL11A with synthetic peptides showed that these enzymes recognize an N-terminal XPK motif. Many eukaryotic proteins have an XPK at their N-terminus and are known to be N-terminally methylated. We hypothesize that Ntm1 and its orthologs are responsible for these modifications. We also identified and characterized a fungal-specific enzyme, ribosomal protein lysine methyltransferase 5 (Rkm5), and showed that it methylates ribosomal protein Rpl1ab at a lysine residue. Rkm5 was able to methylate Rpl1ab bound to ribosomes and a synthetic peptide corresponding to the methylation region on Rpl1ab. We also uncovered a histidine methyltransferase (Hpm1) and showed that it methylates the conserved ribosomal protein Rpl3. This was a novel discovery because it was the first histidine methyltransferase described in the literature and the first report of histidine methylation in yeast. We showed that Hpm1 can only methylate ribosome-associated Rpl3 in mature and nascent ribosomes in the cytoplasm and nucleus, respectively. This was supported by the detection of histidine methylation in ribosome and nuclear fractions in vivo. This indicated a potential role of Hpm1 in ribosome biogenesis and/or translation. Northern blot and polysome profile analyses showed that loss of Hpm1 results in pre-rRNA processing defects and a deficit of 60S large ribosomal subunits, demonstrating that Hpm1 plays a significant role in ribosome production. Loss of Hpm1 also resulted in increased errors during translation elongation, suggesting that it also plays an important role in translation. Analysis of a strain deficient in Rpl3 methylation showed that methylation of Rpl3 is dispensable for ribosome production but essential for accurate translation. This revealed that Hpm1 is a multifunctional enzyme with independent roles in ribosome biogenesis and translation; the latter regulated by Rpl3 methylation and the former by methylation of yet unknown proteins. Similar analyses of all of the additional known ribosomal protein methyltransferases showed that most are involved in ribosome biogenesis and that all are important for translational fidelity

Methylation of yeast ribosomal protein Rpl3 promotes translational elongation fidelity

Rpl3, a highly conserved ribosomal protein, is methylated at histidine 243 by the Hpm1 methyltransferase in Saccharomyces cerevisiae. Histidine 243 lies close to the peptidyl transferase center in a functionally important region of Rpl3 designated as the basic thumb that coordinates the decoding, peptidyl transfer, and translocation steps of translation elongation. Hpm1 was recently implicated in ribosome biogenesis and translation. However, the biological role of methylation of its Rpl3 substrate has not been identified. Here we interrogate the role of Rpl3 methylation at H243 by investigating the functional impact of mutating this histidine residue to alanine (rpl3-H243A). Akin to Hpm1-deficient cells, rpl3-H243A cells accumulate 35S and 23S pre-rRNA precursors to a similar extent, confirming an important role for histidine methylation in pre-rRNA processing. In contrast, Hpm1-deficient cells but not rpl3-H243A mutants show perturbed levels of ribosomal subunits. We show that Hpm1 has multiple substrates in different subcellular fractions, suggesting that methylation of proteins other than Rpl3 may be important for controlling ribosomal subunit levels. Finally, translational fidelity assays demonstrate that like Hpm1-deficient cells, rpl3-H243A mutants have defects in translation elongation resulting in decreased translational accuracy. These data suggest that Rpl3 methylation at H243 is playing a significant role in translation elongation, likely via the basic thumb, but has little impact on ribosomal subunit levels. Hpm1 is therefore a multifunctional methyltransferase with independent roles in ribosome biogenesis and translation

Methylation of yeast ribosomal protein Rpl3 promotes translational elongation fidelity

Rpl3, a highly conserved ribosomal protein, is methylated at histidine 243 by the Hpm1 methyltransferase in Saccharomyces cerevisiae. Histidine 243 lies close to the peptidyl transferase center in a functionally important region of Rpl3 designated as the basic thumb that coordinates the decoding, peptidyl transfer, and translocation steps of translation elongation. Hpm1 was recently implicated in ribosome biogenesis and translation. However, the biological role of methylation of its Rpl3 substrate has not been identified. Here we interrogate the role of Rpl3 methylation at H243 by investigating the functional impact of mutating this histidine residue to alanine (rpl3-H243A). Akin to Hpm1-deficient cells, rpl3-H243A cells accumulate 35S and 23S pre-rRNA precursors to a similar extent, confirming an important role for histidine methylation in pre-rRNA processing. In contrast, Hpm1-deficient cells but not rpl3-H243A mutants show perturbed levels of ribosomal subunits. We show that Hpm1 has multiple substrates in different subcellular fractions, suggesting that methylation of proteins other than Rpl3 may be important for controlling ribosomal subunit levels. Finally, translational fidelity assays demonstrate that like Hpm1-deficient cells, rpl3-H243A mutants have defects in translation elongation resulting in decreased translational accuracy. These data suggest that Rpl3 methylation at H243 is playing a significant role in translation elongation, likely via the basic thumb, but has little impact on ribosomal subunit levels. Hpm1 is therefore a multifunctional methyltransferase with independent roles in ribosome biogenesis and translation

doi:10.1016/j.mrfmmm.2008.08.002

a b s t r a c t RecA is required for recombinational processes and cell survival following UV-induced DNA damage. recA433 is a historically important mutant allele that contains a single amino acid substitution (R243H). This mutation separates the recombination and survival functions of RecA. recA433 mutants remain proficient in recombination as measured by conjugation or transduction, but are hypersensitive to UV-induced DNA damage. The cellular functions carried out by RecA require either recF pathway proteins or recBC pathway proteins to initiate RecA-loading onto the appropriate DNA substrates. In this study, we characterized the ability of recA433 to carry out functions associated with either the recF pathway or recBC pathway. We show that several phenotypic deficiencies exhibited by recA433 mutants are similar to recF mutants but distinct from recBC mutants. In contrast to recBC mutants, recA433 and recF mutants fail to process or resume replication following disruption by UV-induced DNA damage. However, recA433 and recF mutants remain proficient in conjugational recombination and are resistant to formaldehydeinduced protein-DNA crosslinks, functions that are impaired in recBC mutants. The results are consistent with a model in which the recA433 mutation selectively impairs RecA functions associated with the RecF pathway, while retaining the ability to carry out RecBCD pathway-mediated functions. These results are discussed in the context of the recF and recBC pathways and the potential substrates utilized in each case

Histidine Methylation of Yeast Ribosomal Protein Rpl3p Is Required for Proper 60S Subunit Assembly

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein production

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

A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7