Leveraging advancements in next-generation sequencing technology, various epigenetic mechanisms, including DNA methylation, histone acetylation, and mRNA methylation, have been confirmed as significant contributors to numerous physiological and pathological processes, including intrinsic aging [14]. Nevertheless, the precise role of m6A modification in collagen metabolism during extrinsic aging (photoaging) and the underlying regulatory mechanisms of m6A effectors remain poorly understood. In this study, we demonstrate a significant increase in global m6A levels in UVR-induced photoaging models compared to controls, primarily attributed to the upregulation of KIAA1429. Our combined analysis of MeRIP-seq, RNA-seq, and GO analysis reveals that differentially expressed genes (DEGs) involved in collagen metabolism and extracellular processes are significantly regulated by m6A during photoaging. Notably, MFAP4, a key protein in elastic fiber formation and collagen metabolism, is identified as m6A methylated and regulated by KIAA1429. Furthermore, the knockdown of KIAA1429 promotes collagen production in UVR-induced photoaging, an effect attenuated by the depletion of MFAP4.

Initially, we showed that global m6A levels were significantly increased in UVR-induced photoaged mice skin and UVR-irradiated HDFs cells. The results of the cellular fraction are consistent with previous findings reported by Ouyang et al. [15]. However, earlier studies by Xiang and colleagues reported that the m6A level in poly(A)+ RNA was transiently induced and peaked rapidly at 2 min, followed by a gradual decrease after UVC irradiation in U2OS cells [6]. A recent report showed that UVB irradiation decreased the total and poly(A)+ RNA m6A levels in both HaCaT and normal human epidermal keratinocyte (NHEK) cells [7]. It is possible that m6A modification exhibited tissue- and cell-specific regulations [3], since the samples we used were different from other studies. In addition, previous studies have found that different wavelengths of UV light could induce distinct cellular and molecular responses [16], we speculated that this might be also related to the different results in m6A levels.

Cellular m6A modification levels were regulated through the coordinated activity of m6A methylases and demethylases. So, we detected the expression of m6A writers and erasers that have been reported to date and found that KIAA1429 was the most significantly increased writer using multiple assays, which could explain the elevated m6A levels in UVR-induced photoaging. Meanwhile, we have noted that several other writers and erasers were also significantly changed as presented in Fig. 1H, and some of the changes were consistent with altered m6A levels, which cannot exclude that m6A modifications levels are modulated by other regulators in UVA-induced photoaging. Several studies have reported that m6A-related enzymes including METTL14, METTL3 and FTO participated in the regulation of m6A levels after UV irradiation in different samples, of which different biological effects have been demonstrated [6, 7, 15]. Therefore, it remains to be elucidated of other differentially expressed writers and erasers from the current study in the future.

KIAA1429, the largest known protein of the methyltransferase complex, has been recognized as an indispensable factor for the entire process of mRNA methylation since 2015 [17]. The m6A modification region by KIAA1429 was enriched in the 3′-UTR and stop codon of RNA sequences [18]. This corroborates, which is consistent with Fig. 2D of this research. Several studies have reported that KIAA1429 mainly acts as an oncogene in a variety of malignancies, as well as multiple non-neoplastic diseases, indicating its versatility [19, 20]. In addition, a recent study found that the expression of KIAA1429 varied in H2O2-induced senescence cells, replicative senescence cells and young human embryonic lung fibroblasts, but the investigators did not further explore the mechanism of altered KIAA1429 expression on cell senescence [21]. In this study, we revealed that the expression of KIAA1429 remarkably increased in UVR-induced photoaging model. KIAA1429 knockdown decreased the global m6A levels and m6A enrichment in the MFAP4 transcript. Moreover, KIAA1429 negatively regulated collagen production through inhibition of the MFAP4 transcription. These findings support the critical role of KIAA1429 in regulating photoaging through participating in collagen metabolism. Nonetheless, the specific mechanism of the upregulation of KIAA1429 in photoaging remains enigmatic.

We also observed that MFAP4 expression was significantly reduced in UVR-induced photoaged mouse skin and UVR-irradiated HDFs cells. The result was consistent with previous studies [12, 22]. MFAP4, an extracellular matrix protein, manifests an ability to attach itself to collagen, fibrillins, and tropoelastin, thereby playing a crucial role in microfibril assembly, tropoelastin coacervation, and collagen degradation [12, 23]. Moreover, MFAP4 has been found to promote the activation of the TGF-b pathway, which has a key role in tissue remodeling, and seems to be itself a product of TGF-b-related signaling, thus possibly activating an autocrine circuit in several tissue remodeling-associated diseases, such as fibrosis or cardiovascular disorders [24]. In the present study, we found that knockdown of MFAP4 significantly reduced the expression of TGF-bRII, suppressed the signal of colocalization of COL1A2 and MFAP4, and increased the secretion of MMP1 in UV-irradiated KIAA1429 knockdown HDF cells, suggesting that MFAP4 could participate in photoaging through modulating the TGF-β pathway. However, further experiments, such as loss- and gain- of function assays, are warranted to corroborate this finding.

m6A modifications can modulate gene expression through regulating mRNA stability, mRNA export, mRNA translation, etc. [5]. We showed that KIAA1429 knockdown significantly reduced m6A enrichment in the MFAP4 transcript, and m6A RNA methylation levels negatively regulate MFAP4 mRNA expression, accompanied by a decrease in protein level. However, we did not investigate the specific m6A reader protein that recognizes the m6A-modified locus in MFAP4 and the underlying mechanisms that lead to the alterations in mRNA and protein. It is possible that either the m6A reader affected MFAP4 mRNA stability or other biological processes, necessitating further experimentation, such as crosslinking and immunoprecipitation (CLIP) or RNA pull-down technology, to elucidate the precise molecular events contributing to these alterations in gene expression.

GO enrichment analysis has been widely used to investigate the coherent functional signal among multiple genes. For these unique peaks-containing genes in the UVR sample, we found that some of them were highly involved in response to oxidative stress and autophagy processes [25], which have been shown to play an important role in photoaging. Moreover, in relation to genes exhibiting disparities in both m6A levels and expression levels, the outcomes of GO enrichment analysis evinced a multiplicity of biological processes closely associated with extracellular matrix and collagen metabolism within the hyper-down fraction of genes. Conversely, other portions of genes failed to manifest any processes associated with photoaging. These results suggest that m6A modifications are involved in multiple aspects of the photoaging mechanism, not only MFAP4 in this study. Therefore, more investigations on other genes with altered m6A modifications and associated with photoaging are needed in the future.