Dynamic methylome of internal mRNA N7-methylguanosine and its regulatory role in translation

Abstract

Over 150 types of RNA modifications are identified in RNA molecules. Transcriptome profiling is one of the key steps in decoding the epitranscriptomic panorama of these chemical modifications and their potential functions. N⁷-methylguanosine (m⁷G) is one of the most abundant modifications present in tRNA, rRNA and mRNA 5'cap, and has critical roles in regulating RNA processing, metabolism and function. Besides its presence at the cap position in mRNAs, m⁷G is also identified in internal mRNA regions. However, its transcriptome-wide distribution and dynamic regulation within internal mRNA regions remain unknown. Here, we have established m⁷G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m⁷G miCLIP-seq) to specifically detect internal mRNA m⁷G modification. Using this approach, we revealed that m⁷G is enriched at the 5'UTR region and AG-rich contexts, a feature that is well-conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m⁷G modification is dynamically regulated under both H₂O₂ and heat shock treatments, with remarkable accumulations in the CDS and 3'UTR regions, and functions in promoting mRNA translation efficiency. Consistently, a PCNA 3'UTR minigene reporter harboring the native m⁷G modification site displays both enriched m⁷G modification and increased mRNA translation upon H₂O₂ treatment compared to the m⁷G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m⁷G methylome and highlight m⁷G as a novel epitranscriptomic marker with regulatory roles in translation.

Fig. 1

miCLIP-seq provides higher resolution than conventional MeRIP-seq. a Illustration of m⁷G miCLIP-seq. Fragmented RNAs are incubated with anti-m⁷G antibody. After UV cross-linking, covalently bound antibody-RNA complexes are recovered by protein A affinity purification. RNAs are then released by proteinase K digestion and reverse transcribed. During this step, peptide fragments that remain on RNAs lead to cDNA truncations or mutations. The cDNA library is then amplified by PCR and subjected to deep sequencing. b Integrative Genomics Viewer (IGV) tracks showing reads from three different libraries along 18S rRNA. The red arrow represents m⁷G residue at position 1639 of 18S rRNA. c IGV tracks showing reads from miCLIP libraries under 12 different UV conditions along 18S rRNA. d Parameter settings of cross-linking times, wave length and energy input for each UV condition. e IGV tracks showing reads from miCLIP libraries under 12 different UV conditions around m⁷G1639. The red bar along each track represents the truncation reads number at each position

Fig. 2

The transcriptome landscape of m⁷G reveals the enrichment of internal m⁷G near start codon in human mRNAs. a Workflow for mRNA m⁷G miCLIP-seq. b Pie chart showing percentage of mRNA m⁷G clusters in each non overlapping segment in HeLa (left panel) and 293T (right panel). Segments are annotated by Ensembl database (hg38, release 86) and the cap region is defined as 50 nt downstream from the 5′ terminus. c Motif analysis of internal mRNA m⁷G in HeLa (upper panel) and 293T (lower panel) mRNAs by HOMER. d Distribution of internal m⁷G across HeLa (blue) and 293T (red) mRNA segments. Each segment is normalized according to its average length from Ensembl database. e Venn plot showing the numbers of mRNAs displaying internal m⁷G in HeLa and 293T cells. f Bar plot chart showing the significant GO terms for HeLa mRNAs containing internal m⁷G. g Representative mRNAs displaying m⁷G clusters in both HeLa (upper panel) and 293T (lower panel) cells. m⁷G clusters are highlighted with red arrows along the transcripts. Transcript architecture is shown beneath, with thin parts corresponding to UTRs and thicker ones to CDS; exon-exon junctions are indicated by vertical black lines. h Boxplot chart showing increased translation efficiency (TE) for HeLa (upper panel) and 293T (lower panel) mRNAs displaying m⁷G within 5′UTR, CDS or 3′UTR compared to mRNAs without internal m⁷G (Mann–Whitney U test). Translation efficiency results are downloaded from GSE63591 and GSE65778

Fig. 3

m⁷G methylome shows conservation between human and mouse. a Pie chart showing percentage of mRNA m⁷G in each non-overlapping segment in mESCs. Segments are annotated by Ensembl database (mm10, release 68). b Distribution of internal m⁷G across mRNA segments in mESCs. c KEGG pathway analysis of mRNAs displaying internal m⁷G. Pathways are filtered by P < 0.005 and FDR < 0.1 as default. d Two key embryonic mRNAs displaying m⁷G clusters in mESCs. e Pie chart showing percentage of mRNA m⁷G in each non overlapping segment in mouse brain. f Distribution of internal m⁷G across mRNA segments in mouse brain. g KEGG pathway analysis of mRNAs displaying internal m⁷G. Pathways are filtered by P < 0.005 and FDR < 0.1 as default. h Two brain specific mRNAs displaying m⁷G clusters in mouse brain

Fig. 4

Stresses enhance enrichment of internal m⁷G in CDS and 3′UTR in 293T cells. a Barplot chart showing the GO terms for mRNAs displaying oxidative stress-enhanced internal m⁷G (left panel, n = 1924) and heat shock-enhanced internal m⁷G (right panel, n = 2009) in 293T cells. b Barplot chart showing percentage of mRNA m⁷G in each non-overlapping segment under oxidative stress (upper panel) and heat shock (lower panel) in 293T cells. c Numbers of sustained (blue) and heat/oxidation-triggered internal m⁷G (red) across mRNA segments under oxidative stress (upper panel) and heat shock (lower panel) in 293T cells. d Two mRNAs displaying oxidative stress-enhanced internal m⁷G modification in control (blue) and hydrogen peroxide-treated (red) 293T cells. e Two mRNAs displaying heat shock-enhanced internal m⁷G modification in control (blue) and heat shock-treated (red) 293T cells. f Western blot assay showing increased METTL1 protein expression after heat shock treatment. γ-actin serves as a protein loading control. g Quantification of three biological replicates of western blot assays showing increased METTL1 protein expression after heat shock treatment (n = 3). h Dot blot assay showing m⁷G levels of 293T mRNAs under control and heat shock conditions. Methylene blue staining indicates equal RNA loadings. i Quantification of three biological replicates of dot blot assays showing increased m⁷G levels of 293T mRNAs upon heat shock treatment. (n = 3). Data represent mean ± SEM. The P values were calculated by a two-tailed unpaired Student’s t test

Fig. 5

Internal m⁷G promotes PCNA mRNA translation. a IGV tracks displaying miCLIP-seq read distribution along PCNA in control (blue) and hydrogen peroxide-treated (red) 293T cells. b qPCR assay showing m⁷G enrichment of endogenous PCNA mRNA upon H₂O₂ treatment (n = 3). c Western blot assay showing increased PCNA protein expression after H₂O₂ treatment. γ-actin serves as a protein loading control. d Quantification of western blot assays from five biological replicates showing increased PCNA protein expression after H₂O₂ treatment (n = 5). e qRT-PCR analysis of the enrichment of endogenous PCNA mRNAs from the polysome fraction upon H₂O₂ treatment (n = 3). f Reporter constructs of Luciferase-WT-PCNA and Luciferase-MUT-PCNA. A 100 nt fragment containing m⁷G site from 3′UTR of PCNA was inserted after Luciferase CDS. Luciferase-MUT-PCNA was identical to Luciferase-WT-PCNA, except that one guanine (bold red) within the m⁷G motif was mutated to adenosine (bold red). g m⁷G enrichment of exogenous Luciferase-WT-PCNA reporter RNA upon H₂O₂ treatment assayed by qPCR (n = 3). h Dual luciferase assay showing increased translation efficiency of Luciferase-WT-PCNA reporter upon H₂O₂ treatment (n = 3). i Dual luciferase assay showing decreased translation efficiency of Luciferase-MUT-PCNA reporter compared to Luciferase-WT-PCNA reporter in standard condition (n = 3). Data represent mean ± SEM. The P values were calculated by a two-tailed unpaired Student’s t test