The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs

Abstract

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N⁶-methyladenosine (m⁶A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m⁶A in 18S rRNA at position A₁₈₃₂ We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m⁶A in rRNA in stemness, differentiation, development, and diseases.

Keywords: m6A; methyltransferase; pluripotency.

Figure 1.

METTL5 methylates RNA in vitro. (A) Results of screen of METTL proteins for RNA methyltransferase activity. In vitro RNA methyltransferases assays (MTA) with purified recombinant GST-METTL proteins on total RNA, RNA >200 nt, and double-stranded RNA as substrates and tritium-labeled SAM (S-adenosylmethionine) as methyl donor. RNA was purified and tritium incorporation quantified by liquid scintillation counting (LSC). Symbols were assigned based on counts per minute (CPM) of the in vitro reaction relative to the corresponding no-substrate control: (−) Activity less than threefold; (+) threefold to sevenfold; (++) sevenfold to 15-fold; (+++) >15-fold; (ND) substrate–protein pairs not tested. (B) In vitro RNA MTA with recombinant WT GST-METTL5 (left) and GST-METTL8 (right) on total HeLa RNA with tritium-labeled SAM. GST alone and reactions with no RNA substrate were used as controls. CPM for one representative experiment as averages of three technical replicates with standard deviation (SD) is shown. (C) In vitro RNA MTA with GFP-METTL5 purified from HeLa-FRT as in B. (D) In vitro RNA MTA with WT GFP-METTL5 and GFP-METTL5 D81-to-A, D81-to-H, and N126-to-A mutants as in C. Data are displayed as percentage of activity of WT enzyme calculated from an average of two biological and three technical replicates with SD. (E) TRMT122 interacts with METTL5. GFP-METTL5 was affinity-purified (in triplicates) from HeLa-FRT whole-cell extracts and enriched proteins identified by mass spectrometry. Lysates from cells expressing GFP alone were used as control. Data are visualized as a volcano plot. The log₂ fold change (FC) of GFP-METTL5 to control in label-free quantification is plotted against the −log₁₀ of the FDR calculated by a permutation-based FDR-adapted t-test as described before (Ignatova et al. 2019).

Figure 2.

METTL5 catalyzes m⁶A formation in RNA in vitro and in cells. (A) Principle of Methyl-NAIL-MS (Nucleic acid isotope labeling-coupled mass spectrometry) (Reichle et al. 2019). Cells were grown in the presence of CD₃-methionine for metabolic D₃-labeling of methylated nucleosides. Total RNA isolated from these cells was in vitro methylated by recombinant METTL5 with unlabeled SAM. RNA was digested and methylated nucleosides were analyzed by LC-MS/MS. (B) LC-MS/MS elution profiles of indicated modifications in in vitro methylated RNA. In vitro methylated nucleosides (blue) can be distinguished from endogenous methylations (black) by a mass shift of +3 Da. Overlaid MS/MS chromatograms of 6-methyladenosine (m⁶A), 2′-O-methyladenosine (Am), and 5-methylcytosine (m⁵C) are shown. (C, left) Scheme of RNA size exclusion chromatography followed by LC-MS/MS (Chionh et al. 2013). The collected and analyzed fractions are highlighted in gray with sizes of the predominant RNA species per fraction (F1: 28S rRNA; F2: 18S rRNA; F3: 5.8S rRNA and 5S rRNA; F4: 5S rRNA; F5: tRNAs and pri-miRNAs) in the table. (Right) Absolute quantifications of modified nucleosides per respective RNA for 6-methyladenosine (m⁶A), 2′-O-methyladenosine (Am), and 7-methylguanosine (m⁷G) (Borland et al. 2019). Average of three biological replicates and SD are plotted. See Supplemental Figure S2b for additional data. The small decrease in m⁶A abundance in fraction F1 could be due to a minor contamination with 18S rRNA as suggested by model from Piekna-Przybylska et al. (2008) (see also https://people.biochem.umass.edu/fournierlab/3dmodmap/humssu2dframes.php). (E) In vitro RNA MTA with recombinant GFP-METTL5 on 18S rRNA isolated from WT and Mettl5 KO mESC with tritium-labeled SAM. Tritium signal was quantified by liquid scintillation counting (LSC). CPMs for one representative experiment as averages of three technical replicates with SD are plotted. (****) P < 0.0001; (*****) P < 0.00001, calculated with Welch's test (unpaired t-test).

Figure 3.

Mettl5 KO mESCs exhibit reduced pluripotency and differentiation defects. (A) Representative bright field images of WT and Mettl5 KO mESCs (clones C9, F8, and G2) after 6 d in serum LIF medium. (B) Representative alkaline phosphatase stainings of WT and Mettl5 KO mESCs (clones C9, F8, and G2). (C) Results of gene ontology (GO) analysis of differentially expressed genes in Mettl5 KO (clones C9) compared with WT mESCs. Normalized enrichment scores for the top GO terms with BH-adjusted P-values < 0.05 are plotted. (D) RT-qPCR analysis of the expression levels of pluripotency, gastrulation and lineage marker genes in WT (blue) and Mettl5 KO mESCs (red) after 6 d in Serum LIF media. Fold changes quantified relative (RQ) to WT are plotted. Error bars indicate the standard error on the average RQ-values of three independent KO clones (C9, F8, and G2) and three replicates of WT. (E) Boxplots showing quantification of immunostainings for pluripotency markers NANOG and KLF4 for two independent Mettl5 KO clones (F8 and G2) and WT mESCs. Each dot represents the mean fluorescence intensity of an individual cell. Welch two sample t-test P-values are shown. (F) Cell cycle distributions of Mettl5 KO and WT mESCs analyzed by flow cytometry after staining with propidium iodide (PI). The experiment was performed in three biological replicates. Representative distributions of PI intensities are shown. (G) Schematic representation of embryonic body formation assay (left) and neuronal differentiation protocol (right) via cellular aggregates (CAs). For details, see the Materials and Methods. (H) RT-qPCR analysis of the expression levels of lineage marker genes in WT (blue) and Mettl5 KO mESCs (red) at day 8 of the embryonic body (EB) formation assay. Fold changes quantified relative (RQ) to WT are plotted. Error bars indicate the standard error on the average RQ values of three independent KO clones (C9, F8, and G2) and three replicates of WT. (I) Immunostaining for the neuronal marker MAP2 in three Mettl5 KO clones (C9, F8, and G2) and WT control neuronal precursor cells (NPCs). Scale bar, 50 µM. DAPI staining (left) and merge (right) are shown.

Figure 4.

Translation is altered in Mettl5 KO cells. (A) Polysome analysis of WT and Mettl5 KO (clone C9) mESCs. Mettl5 KO cells show increased monosome and decreased polysome profiles in sucrose gradients. The experiment was performed in three biological replicates. Data from one representative experiment is shown. (B) OPP-incorporation analysis to measure nascent translation rates in Mettl5 KO and WT mESCs (top panel) and Mettl5 KO and WT MEFs (passage 4) (bottom panel). OPP-Alexa 594 intensities of individual cells were measured by flow cytometry. Medians of OPP-Alexa 594 signal intensities from all replicates (normalized to WT) are displayed as box plots (left) and one representative OPP-Alexa 594 fluorescence intensity distribution per cell line is shown (right). Experiments were performed in two biological and at least two technical replicates. Welch two-sample t-test P-values are shown. (C) Genome-wide changes in transcript abundance and ribosome occupancy in Mettl5 KO (clone C9) and WT mESCs. Heat map displays changes in transcript abundance (RNA, left panel) or ribosome occupancy (RBF, right panel), expressed as log₂ fold change (FC) relative to wild type in the range from −4 to 4, split into four groups: RBF log₂ FC < −0.3 and RNA log₂ FC > 0; RBF and RNA log₂ FC < −0.3; RBF and RNA log₂ FC > 0.3; RBF log₂ FC > 0.3 and RNA log₂ FC < 0. For fold changes, groups and transcript names see Supplemental Table S4. (D) Results of gene ontology (GO) by over-representation analysis of transcripts with decreased ribosomes occupancy (log₂ FC < −0.3), but without reduction at the transcript level (log₂ FC > 0) in Mettl5 KO compared with WT mESCs. Enrichment ratios and BH adjusted P-values are indicated.

Figure 5.

Mettl5 KO mice are subviable and display multiple phenotypic aberrations, including behavioral defects. (A) Mettl5 KO (−/−) mice are subviable; <12.5% of born and weaned mice were knockout animals. The total number of Mettl5 offspring born and weaned: 156 animals. Distribution of genotypes: 57 WT control animals (36.5%), 80 heterozygous (+/−) (51.3%), and 19 homozygous KO mutants (12.2%). From the 19 KO mice, three were females (16%) and 16 males (84%). (B) Weight monitoring of mice from the age of 5 to 15 wk showed significant differences (P = 0.001) between male Mettl5 KO (−/−) homozygous, heterozygous, and control animals (data are means ± SD, n = 23 WT^+/+, n = 10 Mettl5^+/−, n = 10 Mettl5^−/−) (Supplemental Table S5a; Supplemental Fig. S6b for females). (C) Snout deviation was observed in 14-wk-old Mettl5 homozygous KO mice. Micro-CT (top panel) and X-ray (bottom panel) imaging analyses show nasal bones (indicated with white arrowheads in bottom panel) with abnormal growth pattern in seven out of 15 Mettl5 KO males and one out of three KO females. Also, frontal bone suture fusion was incomplete in three out of five Mettl5 KO males (black arrowhead in top panel). (D) Optical coherence tomography (OCT) images of retinas show abnormal retrolental (red X) tissue present in the eyes of 16-wk-old Mettl5 KO mice, due to alterations in hyaloid vascular system regression. This phenotype was observed in six out of eight Mettl5 KO males and three out of three KO females. The right eye was more affected (L- and R-labeled images). Normal hyaloid regression in WT mice is also displayed. (E) Hematoxylin and eosin staining of testis sections revealed scattered degeneration in the seminiferous tubules of four out of eight Mettl5 KO 16-wk-old males. Scale bars, 250 µm and 50 µm. (F) Eight-week-old Mettl5 KO mice were hypoactive (left) and hypoexploratory (right) compared with WT control mice during a 20-min open field test (Garrett et al. 2012). (***) P < 0.001 genotype effect with repeated measures ANOVA. Data are means ± SEM, males and females pooled, n = 45 WT, n = 13 Mettl5 KO (Supplemental Table S6). (G) Diseases and phenotypes described to be associated with METTL5 sequence variants in human and mice as well as in a zebrafish METTL5 morpholino model.