Dataset Information

RPL40 methyltransferase discovery and characterisation

ABSTRACT: The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here we have identified SMYD5 as a ribosomal protein methyltransferase that catalyses trimethylation of RPL40 at lysine 22. Through a systematic mass spectrometry-based approach, we show that the human ribosome has 12 primary sites of protein methylation, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity towards RPL40 K22 in vitro, and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decrease polysome levels. By systematic analysis of its recognition motif, we show that SMYD5 requires a tyrosine in the +2 position, and thereby is incapable of methylating its previously reported histone substrates.

ORGANISM(S): Homo Sapiens

SUBMITTER: Joshua Hamey

PROVIDER: PXD056489 | panorama | Wed Apr 16 00:00:00 BST 2025

REPOSITORIES: PanoramaPublic

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SMYD5 is a ribosomal methyltransferase that trimethylates RPL40 lysine 22 through recognition of a KXY motif.

Hamey Joshua J JJ Shah Manan M Wade John D JD Bartolec Tara K TK Wettenhall Richard E H REH Quinlan Kate G R KGR Williamson Nicholas A NA Wilkins Marc R MR

Cell reports 20250405 4

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here, we identify SET and MYND domain-containing protein 5 (SMYD5) as a ribosomal protein methyltransferase that catalyzes trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we identify 12 primary sites of protein methylation in ribosomes from K562 cells, including at RPL40 K22. Through in vitro methylation of synthetic R ...[more]

PMID: 40184250

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Project description:Eukaryotic ribosomal RNA carries diverse posttranscriptional modifications, among which the evolutionarily conserved 2’-O-methylation (2’-O-Me) occurs at more than 100 sites and is essential for ribosome biogenesis. Plasticity of 2´-O-Me in ribosomes and its functional consequences in human disease are not yet known. Here, we present the full rRNA 2’-O-Me landscape (ribomethylome) of human acute myeloid leukemia (AML) through profiling 94 patient samples as well as 21 normal hematopoietic samples of 5 different lineages. While interior modification sites in functional centers are persistently fully 2’-O-methylated in human AMLs, methylation on ribosome exterior sites is unprecedentedly dynamic. Higher 2’-O-methylation on exterior dynamic sites is associated with leukemia stem cell (LSC) signatures. Forced expression of enzymatically active but not of the catalytic defect 2’-O-methyltransferase FBL induces AML stemness and accelerates leukemogenesis in patient-derived xenografts. Mechanistically, ribomethylome dynamics shifted mRNA ribosome translation preferences. High rRNA 2’-O-Me enhances translation of amino acid transporters enriched in optimal codons and subsequently increases intra-cellular amino acid levels. Methylation on a single exterior modification site affects leukemia stem cell activity. The Guanosine 1447 on the small subunit ribosomal RNA is the most variable site in primary AMLs. Gm1447 is increased in leukemia stem cell populations compared to non-leukemogenic blast cells and AML specimens with higher Gm1447 are enriched for leukemia stem cell genes. Comparison of Gm1447high and Gm1447low ribosome structure solved by cryo-electron microscopy demonstrated disassociation of LYAR from Gm1447low ribosomes. Suppression of Gm1447 alone is sufficient to suppress translation of amino acid transporters, resulting in decreased cellular amino acid levels and leukemia stem cell activity. Taken together, our data reveal the dynamic FBL-mediated rRNA 2'-O-Me landscape as a novel epitranscriptomic level of control employed by leukemic stem cells and may enable new strategies to target human AML.

Dataset Information

RPL40 methyltransferase discovery and characterisation

Publications

SMYD5 is a ribosomal methyltransferase that trimethylates RPL40 lysine 22 through recognition of a KXY motif.

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