m6A mRNA Methylation Decorates Genes Involved in Fibrinogen Synthesis to Regulate Liver Regeneration in Mice
The role of m6A RNA methylation in liver regeneration is unclear. This study aimed to determine the role of m6A methylation in liver regeneration after a 70% hepatectomy (HEPA) using liver specific Mettl 14 KO male mice. Analysis was conducted on post-operative days 1, 3, or 7 (HEPA1, 3, or 7) in control (FLOX) mice. In Flox mice, CyclinD1 protein expression was highest on postoperative day 3 (HEPA3) consistent with a dynamic increase in hepatocyte replication. The abundance of Mettl14 protein presented a similar pattern on HEPA3. Then, we performed hepatectomy in Mettl14 KOs (M14KO) and Flox controls and observed significantly higher post-surgical mortality in mutants. In Flox mice, CyclinD1 protein levels and Ki67 were markedly increased on HEPA3 compared to SHAM, while being downregulated in M14KO. Characterizing the m6A epitranscriptomic changes in Flox mice after hepatectomy and contrasting them to hepatectomy in M14KO in HEPA3 revealed enrichment for gene ontology terms associated with endoplasmic reticulum, inflammation, and apoptosis. Differentially methylated genes in M14KO compared to Flox on HEPA3 were also enriched for PPAR and AMPK signaling. Finally, we identified hypomethylated transcripts involved in fibrinogen synthesis, such as Fga, Fgb, and Fgg, by comparing differentially m6A-decorated genes in M14KO versus Flox on HEPA3. Knockdown of fibrinogen leads to suppression of proliferation via activation of p21 protein in AML12 cells. Together, m6A RNA methylation decorates genes involved in fibrinogen synthesis pointing to an important role in liver regeneration consistent with recent work demonstrating the significance of fibrinogen in liver regeneration. Overall design: Four groups of mice: HEPA3-FLOX, HEPA3-Mettl14KO, SHAM3-FLOX, SHAM3-Mettl14KO with n=4 per group, where each sample was run as input (usual bulk RNA-seq) and with IP using anti-m6A antibody as m6A.