Skip to main content
Open Access Publications from the University of California


UCLA Electronic Theses and Dissertations bannerUCLA

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

  • Author(s): Al-Hadid, Qais K.
  • Advisor(s): Clarke, Steven G
  • et al.

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.

Main Content
Current View