背景:N6-甲基腺苷(m6A)是真核mRNAs中最丰富和可逆的修饰,然而,其在哺乳动物表皮发育中的功能仍未完全阐明。
目的:探讨METTL14(甲基转移酶14)的作用,m6A甲基转移酶之一,维持表皮稳态。
方法:我们构建了表皮基底细胞中Mettl14失活的小鼠。通过H&E染色和免疫荧光染色探索表型。为了探索潜在的机制,我们进行了RNA-seq,野生型和Mettl14失活表皮角质形成细胞的核糖体谱分析和MeRIP-seq。此外,将HaCaT细胞用于体外验证。
结果:小鼠表皮中Mettl14的失活导致表皮短暂增厚和表皮干细胞池耗尽。有趣的是,我们发现XVII型胶原蛋白(Col17a1)的mRNA,整合素β4(Itgβ4)和α6(Itgα6)具有m6A修饰,Mettl14灭活表皮中的蛋白质表达降低。此外,在表皮特异性Mettl4灭活小鼠中,表皮从真皮脱离,并呈现类似于交界性大疱性表皮松解症(JEB)的表型,这可能是由于半染色体损害(COL17A1、ITGB4和ITGA6降低)所致。在HaCaT细胞中敲除Mettl14会损害自我更新,并降低COL17A1,ITGB4和ITGA6的蛋白质水平,而Itgβ4敲除会抑制集落形成。
结论:我们的研究强调了METTL14在维持表皮稳态中的作用,并通过m6A介导的Col17a1,Itgβ4和Itgα6的翻译抑制确定了其关键作用。我们的研究表明,METTL14可能是治疗半球体缺乏疾病的潜在治疗靶点。比如JEB。
BACKGROUND: N6-methyladenosine (m6A) is the most abundant and reversible modification occurring in eukaryotic mRNAs, however, its functions in mammalian epidermal development are still not fully elucidated.
OBJECTIVE: To explore the role of METTL14 (Methyltransferase like 14), one of the m6A methyltransferases, in maintaining epidermal homeostasis.
METHODS: We constructed mice with Mettl14-inactivation in the epidermal basal cells. The phenotype was explored by H&E staining and immunofluorescence staining. To explore the underlying mechanisms, we performed RNA-seq, Ribosome profiling and MeRIP-seq on wild-type and Mettl14-inactivation epidermal keratinocytes. Moreover, HaCaT cells were used for in vitro validation.
RESULTS: Inactivation of Mettl14 in murine epidermis led to transient thicker epidermis and exhaustion of the epidermal stem cell pool. Interestingly, we found that the mRNA of type XVII collagen (Col17a1), integrin β4 (Itgβ4) and α6 (Itgα6) had m6A modifications, and the proteins expression were decreased in Mettl14-inactivated epidermis. Furthermore, in epidermis-specific Mettl4-inactivated mice, the epidermis was detached from the dermis and presented a phenotype similar to junctional epidermolysis bullosa (JEB), which may result from hemidesmosomes damage (decrease of COL17A1, ITGB4 and ITGA6). Knockdown of Mettl14 in HaCaT cells impaired the self-renewal and decreased the protein level of COL17A1, ITGB4 and ITGA6 and Itgβ4 knockdown inhibited colony formation.
CONCLUSIONS: Our study highlighted the role of METTL14 in the maintenance of epidermal homeostasis and identified its critical role through m6A-mediated translational inhibition of Col17a1, Itgβ4 and Itgα6. Our study suggested that METTL14 may be a potential therapeutic target for the treatment of hemidesmosomes-deficient diseases, such as JEB.