Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation

Abstract

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N⁷-methylguanosine (m⁷G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m⁷G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m⁷G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m⁷G tRNA methylome and highlights its essential role in ESCs with links to human disease.

Keywords: MeRIP-seq; Mettl1; N(7)-methylguanosine; RNA methylation; TRAC-seq; Wdr4; embryonic stem cells; m(7)G; neural development; tRNA; translation.

Figure 1. Global profiling of tRNA m⁷G modifications by m⁷G MeRIP-Seq

(A) Western blot of the control and Mettl1 KO samples with indicated antibodies. (B) Anti-m⁷G Northwestern blot of m⁷G modifications (Upper panel). RNA samples were separated on a TBE-urea gel, then transferred to nylon membrane for Western blotting using anti-m⁷G antibody. Northern blot of U6 snRNA was used a loading control (Lower panel). (C) Western blot of the METTL1 rescue samples with indicated antibodies. (D) Anti-m⁷G Northwestern blot of m⁷G modifications (Upper panel) and Northern blot of U6 snRNA (Lower panel). (E) Small RNA Northern blot of the anti-m⁷G IP products with indicated probes. (F) Schematic diagram of small RNA m⁷G MeRIP-seq to identify the m⁷G methylome. Small RNAs were isolated and treated with Alkb to remove modifications and then used for cDNA library construction. (G) Population of small RNA mapping in m⁷G MeRIP-Seq. Samples without Alkb treatment (Alkb-) were used as controls. n=2. (H) Scatter plot of tRNA relative enrichment of tRNA in m⁷G MeRIP-Seq. Relative enrichment was calculated by comparing the IP/Input ratio in the WT samples to the KO samples. (I) List of m⁷G modified tRNAs identified by m⁷G MeRIP-Seq. (J) Confirmation of m⁷G modified tRNAs by anti-m⁷G IP and Northern blot with indicated probes. No m⁷G modification was identified in GlnCTG, which was used as a control.

Figure 2. Single nucleotide resolution mapping of m⁷G modifications by m⁷G TRAC-Seq

(A) Schematic diagram of tRNA reduction and cleavage at m⁷G sites. RNA samples were treated with NaBH₄ and aniline to induce the site-specific cleavage of RNA at m⁷G modified guanosines. (B) Northern blot of the chemical treated RNA samples with indicated probes. (C) Schematic diagram of the m⁷G TRAC-Seq. (D) Representative images of reads alignment to indicated tRNAs in the Integrative Genomics Viewer (IGV) using m⁷G TRAC-Seq data. Chemical treatment resulted in a site specific decrease of reads at m⁷G modified position. n=2. (E) Schematic diagram of the method to calculate the cleavage score at individual positions using TRAC-Seq data. (F) Representative images of cleavage scores of indicated tRNAs. Pictures show a specific increase of cleavage score at the m⁷G sites. (G) Overlap of m⁷G modified tRNAs identified in m⁷G MeRIP-Seq and m⁷G TRAC-Seq.

Figure 3. Evolutionary sequence conservation and function of m⁷G tRNA modifications

(A) Sequence motif in the m⁷G sites identified by m⁷G TRAC-Seq in mouse cells. (B) Sequences of Gly-tRNAs in mouse and yeast. GlyAAC tRNA exists in mouse but not in the yeast. Green box labels the anticodon and red box indicates the m⁷G motif. (C) Sequences of ArgTCT and AsnGTT tRNAs in different species. Green box labels the anticodon and red box indicates the m⁷G motif. (D) Scatter plot of tRNA expression levels in the Mettl1 KO and control cells. Each dot represents one tRNA genes. x- axis indicates the changes of tRNA expression levels, y-axis indicates p-value. (E) Mann-Whitney test on the expression of the m⁷G and non-m⁷G tRNAs. (F) Expression profile of the 22 m⁷G modified tRNAs. Each box plot shows the expression of a tRNA type that was calculated from the combined expression of all the tRNA genes for the same tRNA type. (G) Small RNA Northern blot of the METTL1 rescue samples with indicated probes. U6 snRNA was used as a loading control.

Figure 4. Mettl1 depletion causes ribosome pausing at m⁷G tRNA-dependent codon containing cell cycle and disease genes

(A) Polysome profiling of the Mettl1 KO and WT cells. (B) Scatter plot of translation efficiency (TE) in the Mettl1 KO and WT cells. TE was calculated by dividing the ribosome protected fragments (RPF) signals to the input RNA-Seq signals. (C) Codon frequency in the CDS region of the genes with increased TE (up), decreased TE (down) and other genes (non) in the Mettl1 KO cells. (D) Ribosome occupancy at individual codon at A sites and A+1 sites. Data were presented as the relative ribosome footprinting signals from Mettl1 KO and WT cells. The codons are separated into four groups based on the modification and expression status of their corresponding tRNAs: codons decoded the m⁷G tRNAs (red); codons decoded by m⁷G tRNAs by wobble effect because of the not detected levels of their corresponding tRNAs (pink); codons decoded by non-m⁷G tRNAs by wobble effect because of the not detected levels of their corresponding tRNAs (grey); codons decoded by non-m⁷G tRNAs (black). (E) Gene ontology analysis of function enrichment in human phenotype and biological process using the TE down-regulated genes upon Mettl1 KO. (F-G) Cell cycle analysis of Mettl1 KO and WT cells. n=3, *p<0.05, **p<0.01.

Figure 5. Mettl1 is required for mESCs self-renewal

(A) qRT-PCR analysis of mRNA expression in Mettl1 KO and control cells with indicated primers. Error bars = mean ±SEM, n=3. (B) Western blot with indicated antibodies. (C) Cellular proliferation in the Mettl1 KO and control cells. 10⁵ cells were seeded in 100 mm tissue culture dishes and then the cell numbers were counted at Day 3 and Day 5. n=3. (D-F) Colony formation assay of Mettl1 KO and control cells. 500 cells were seeded in 6 well plate, one week later, the colony size and number were measured. (D) The representative images of the colonies. Scale bar, 50uM. (E) Colony diameters. n=8, ***p<0.001. (F) Colony numbers. n=6, **p<0.01, ***p<0.001. (G-H) AP staining of the Mettl1 KO and control cells. Scale bar, 100uM. (G) Representative images; (H) Percentage of AP positive colonies. n=10, *p<0.05, ***p<0.001. (I) Scatter plot of mRNA expression using the RNA-Seq data from the Mettl1 KO and control cells. (J-K) Gene ontology analysis of the up-regulated and down-regulated genes upon Mettl1 knockout in mESCs. (L-M) q-RT-PCR analysis of mRNA expression in Mettl1 KO and control cells with indicated primers. Error bars = mean ±SEM, n=3.

Figure 6. Mettl1 knockout disrupts balance of ESC differentiation and impairs neural lineage differentiation

(A) Upper panel: schematic diagram of ESC embryoid body differentiation procedure. Lower panel: qRT-PCR analysis of mRNA expression in Mettl1 KO and control cells (after differentiation for 6 days) with indicated primers. Error bars = mean ±SEM, n=3. (B) Scatter plot of mRNA expression using the RNA-Seq data from the Mettl1 KO and control embryonic bodies (differentiated for 6 days). (C) Gene ontology analysis of functional enrichment in biological process using the differentially expression genes between the Day6 Mettl1 KO and control embryoid bodies. (D) Upper panel: schematic diagram of ESC neural lineage differentiation procedure. Lower panel: qRT-PCR analysis of mRNA expression in Mettl1 KO and control cells (after differentiation for 6 days) with indicated primers. Error bars = mean ±SEM, n=3. (E) Western blot of the METTL1 rescue samples with indicated antibodies. (F) qRT-PCR analysis of mRNA expression in METTL1 rescue samples (after differentiation for 6 days) with indicated primers. Error bars = mean ±SEM, n=3.

Figure 7. Wdr4 knockdown abolishes m⁷G tRNA modification and impairs ESC proliferation and neural differentiation

(A) Schematic diagram of CRISPR mediated knockout of Wdr4 expression in mESCs. (B) PCR to confirm the knockout of Wdr4 in mESCs using genome DNA samples from the knockout and control cells. (C) qRT-PCR analysis of Wdr4 mRNA expression in knockout and control cells. Error bars = mean ±SEM, n=3. (D) Anti-m⁷G Northwestern blot of m⁷G modifications. RNA samples were separated with TBE-UREA gel, when transferred to nylon membrane for Western blotting using anti-m⁷G antibody. Northern blot of U6 snRNA was used a loading control. (E) Small RNA Northern blot of the anti-m⁷G IP products with indicated probes. (F) Northern blot of the chemical treated RNA samples with indicated probes. (G) Cellular proliferation in the Wdr4 and control cells. 10⁵ cells were seeded in 100 mm tissue culture dishes and then the cell numbers were counted at Day 3 and Day 5. (H) Upper panel: schematic diagram of ESC neural lineage differentiation procedure. Lower panel: qRT-PCR analysis of mRNA expression in Mettl1 KO and control cells (after differentiation for 6 days) with indicated primers. Error bars = mean ±SEM, n=3.