Regulation of Methylase METTL3 on Fat Deposition.

N6-methyladenosine (m6A) is the most prevalent and abundant type of internal post-transcriptional RNA modification in eukaryotic cells. METTL3 is a methylation modifying enzyme, which can directly or indirectly affect biological processes, such as RNA degradation, translation and splicing. In addition, it was found that 67% of 3'-UTR regions containing m6A sites had at least one miRNA binding site, and the number of m6A at 3'-UTR sites was closely related to the binding sites of miRNA. With the improvement of human living standards, obesity has become a very serious and urgent problem. The essence of obesity is the accumulation of excess fat. Exploring the origin and development mechanisms of adipocyte from the perspective of fat deposition has always been a hotspot in the field of adipocyte research. The aim of the present review is to focus on METTL3 regulating fat deposition through mRNA/adipocyte differentiation axis and pri-miRNA/pre-miRNA/target genes/adipocyte differentiation and to provide a theoretical basis according to the currently available literature for further exploring this association. This review may provide new insights for obesity, fat deposition disease and molecular breeding.

Introduction

In the early 1970s, a novel RNA epigenetic modification, N6-methyladenosine (m6A), was first discovered and proposed in eukaryotic messenger RNA (mRNA) from Novikoff hepatoma cells.1 N6-methyladenosine (m6A) is one of the most abundant internal modifications in eukaryotic messenger RNA that affects a variety of cellular biological processes, including splicing, processing, nuclear export, stability and decay, translation, cellular differentiation and metabolism.2,3 M6A modification refers to the methylation of the 6th N of adenine on mRNA under the action of methyltransferase complex (MTC), which is a dynamic and reversible process regulated by both methyltransferase and dimethyl transferase,4 such as methyltransferase like 3 (METTL3) and methyltransferase like 4 (METTL4).5,6

METTL3 was discovered and named from Hela cells in 19947 and was conserved from yeast to human, including leading spiral structure LH, nuclear localization signal NLS, Methyltransferase domain MTD containing SAM binding domain and zinc finger motif ZFD.6,8 Studies have shown that zinc finger participates in RNA binding, ZNF1 interacts with RNA electrostatically, whereas ZNF2 interacts with RNA hydrophobically, which suggests that zinc finger is responsible for specifically recognizing RNA and making METTL3 play a role.9 The formation of miRNA requires the cutting of the complex composed of DGCR8 and DROSHA, and METTL3 deletion reduced the binding of DGCR8 to pri-miRNA.10 According to its function, RNA can be divided into two broad categories, including noncoding RNA and encoding protein mRNA. MiRNAs are a group of conservative, small and non-coding RNAs inhibiting translation of or degrading target mRNAs by binding to the complementary sequences in the 3’untranslated region.11 It has been proved that miRNAs play important roles in energy homeostasis,12 sugar and lipid metabolism,13 insulin secretion,14 pancreatic β-cell development,15 and adipocyte differentiation.16 More and more studies have shown that miRNA can interact with transcription factors and important signal molecules related to adipocyte differentiation.17 Adipose tissue deposition is characterized by increased cell size (hypertrophy) and increased cell numbers (hyperplasia) at the cellular level, which indicates that cell differentiation is a necessary process of fat deposition. In addition, studies found the formation of METTL3 and METTL14 heterodimers played an important role in adipocyte differentiation.18 Another study also found METTL3 regulates adipocyte differentiation by regulating genes alone.19 So, METTL3 plays an important role in fat deposition. However, the specific mechanism by which METTL3 regulates fat deposition remains unclear.

Despite recent progress in METTL3 research, the presence and functionality of METTL3 remains largely unknown. Recent studies have reported the emerging roles of METTL3 in the development of fat deposition. The present review focuses on the latest progress in made METTL3 research and provides an up-to-date summary of the association between METTL3 and fat deposition, which may provide insight into METTL3-related molecular biomarkers and increase of fat deposition in animals.

M6A Methylation

Epitranscriptomic m6A modification is dynamically and reversibly regulated by modulators characterized as dedicated demethylases (erasers), m6A binding protein (readers) and methyltransferases (writers), according to their functions,20 Erasers (FTO, ALKBH5) and writers (METTL3, METTL14, WTAP) are responsible for catalyzing and removing m6A, respectively.21–24 In complex METTL3/METTL14/ WTAP, METTL3 and METTL14 form a heterodimer complex and interact with WTAP. METTL3 is identified as a SAM-binding component of the complex and has its own catalytic ability, which is highly conserved in eukaryotes.25 It has been reported that m6A can label pri-miRNAs and identify DGCR8 molecules by METTL3/m6A, participating in the mature process of miRNAs and leading to differential expression of miRNAs in many biological processes.10,26 In addition, it was found that METTL3 knockout decreased the binding activity between DGCR8 and pri-miRNA, leading to decreased expression of mature miRNAs.10 So, understanding the structure of METTL3 and its interaction mechanism with target RNA will help to further understand the post transcriptional regulation level of genetic information.

METTL3 Promoted the Transformation of pri-miRNA into Mature miRNA (miR-21, miR-25, miR-34a, miR126, miR-143-3p, miR-221/222 and miR-320)

MicroRNAs (miRNAs) are a group of single-stranded, non-coding small RNAs that are broadly present in eukaryotic cells and are highly conserved during evolution with a length of 19–24nt.27 As miRNAs are critical in development, differentiation, and fat deposition, their mature are controlled by multiple ways during their biogenesis cascade. Figure 1 shows the role of METTL3 in miRNA maturation. Alarcon et al demonstrated that m6A modification could mark pri-miRNA for processing by recognizing DGCR8 in a METTL3-dependent manner,10 indicating that altered METTL3 mediated m6A modification might be responsible for the aberrant expression of miRNAs in many biological processes. In addition, it was shown that depletion of METTL3 leads to decreased accumulation of miRNAs and to an overaccumulation of pri-miRNAs due to their impaired processing.10,28 Similar to previous results,1 miR-21 was up-regulated when METTL3 was overexpressed.10,29 METTL3-dependent m6A methylation promoted primary miR-34a (pri-miR34a)30 and miRNA-126 (pri-miR126)31 maturation through DGCR8. Other researchers have demonstrated that upregulation of METTL3/m6A modification promotes pri-miR-25,32 pri-miR-221/22233 and pri-miR-143-3p34 maturation (decreasing the expression of pri-miRNA but increasing the expression of pre-miRNA and miRNA). In addition, pre-miR-320 was much less enriched after METTL3 inhibition, indicating that pre-miR-320 was a target of METTL3.35

Figure 1

METTL3 regulates fat deposition by coding RNA and non-coding RNA.

MiRNA (miR-21, miR-25, miR-34a, miR126, miR-143-3p, miR-221/222 and miR-320) Regulated Adipocyte Differentiation by Targeting Target Genes

MicroRNAs (miRNAs), a novel class of endogenous, noncoding, single-stranded RNAs, have emerged as a group of important regulators via degradation or translational inhibition of their target mRNAs.36 As shown in Table 1, miRNA regulates adipocyte differentiation by targeting multiple target genes.

Table 1

METTL3 Regulates Adipocyte Differentiation Through Target Genes

miRNA	Promoting Targeted Genes	Inhibitory Target Gene	Regulation of Target Genes on Adipocyte Differentiation
miR-21		PTEN84	Down regulate85
		SMAD786	Down regulate43
miR-25		BTG287	Down regulate88
		LATS247	Up regulate50
		PTEN89	Down regulate
		FBXW790	Down regulate49
		KLF444	Up regulate44
		C/EBPα44	Up regulate44
miR-34a		AMPK55	Down regulate91
		SIRT153	Down regulate92
		HDAC193	Up regulate94
mir-126		VEGF95	Down regulate65
		IGF-196	Up regulate97
	CRK98		Up regulate62
		IRS-199	Up regulate100
	AKT101		Up regulate102
		PI3K103	Up regulate78
mir-143-3p		MAPK773	Down regulate73
		MAP3K768	Up regulate74
		AKT71	Up regulate
		KLF570	Up regulate76
		PI3K71	Up regulate
		EZH272	Up regulate104
mir-221/mir-222		PTEN105	Down regulate
		TIMP379	Down regulate106
	AKT107		Up regulate
		p27Kip182	Down regulate108
mir-320		ERK1/2109	Down regulate110
		PI3K111	Up regulate
		adipoR1112	Up regulate113

MiR-21 Regulated Adipocyte Differentiation

It was found that nearly 25% of miRNA targets are conserved in the 3’ noncoding region of human, mouse and rabbit.37 In addition, highly conserved miR-21 recognition elements were found in the analysis of PTEN 3’UTR of different species, indicating that PTEN can be combined with mir-21.38 At the same time, the results presented here indicated that the potential signal pathway of miR-21 protection might be achieved by targeting PTEN/AKT signaling pathway. PTEN was the main regulator of the PI3K signaling pathway, which was involved in lipid metabolism and glucose transport in 3 T3-L1 adipocytes.39 Previous studies have also shown that endogenous PTEN expression is down-regulated during 3T3-L1 differentiation,40 and knockdown of PTEN potentiated the increase in insulin-mediated phosphorylation of AKT/ERK and promoted adipogenesis of 3T3-L1 cells.41 The study also indicated that miR-21 directly targets the 3′-UTRs of SMAD7, and negatively regulates mRNA and protein expression levels.42 In addition, SMAD7 regulated 3T3-L1 preadipocyte differentiation and adipogenesis through TGFβ/SMAD and WNT signaling pathway.43

MiR-25 Regulated Adipocyte Differentiation

MiR-25, a member of miR-106b-25 cluster, was significantly downregulated during the differentiation from 3T3-L1 preadipocytes towards mature adipocytes.44 In addition, this study confirmed BTG2,45 FBXW7,46 LATS247 and PTEN48 are targets of miR-25 and had binding sites with miR-25 in the 3’-UTR. Further experiments demonstrated that miR-25 Suppresses 3T3-L1 Adipogenesis by directly targeting KLF4 and C/EBPα.44 FBXW7 inhibits C/EBPα-dependent transcription and inactivation of FBXW7 results in the accumulation of C/EBPα.49 LATS2 regulates the balance between proliferation and differentiation during adipose development. Interestingly, studies provided evidence that LATS2 not only negatively modulates cell proliferation but also positively regulates cell differentiation.50 In addition, a recent study showed that BTG2 downregulates interleukin-6 expression by inhibiting the signal transducer and activator of transcription 3 (STAT3) signaling pathway, which is known to regulate adipocyte differentiation.51 So, miR-25 can regulate adipocyte differentiation through multiple pathways.

MiR-34a Regulated Adipocyte Differentiation

In recent years, reports on miR-34a in human fat have found that miR-34a can target PPARα Gene regulation of human fat deposition in liver,52 which indicated miR-34a played an important role in fat deposition. miR‑34a was revealed to directly target SIRT1 by binding to its 3’‑untranslated region53 and SIRT1 can promote fat mobilization in white adipocytes by repressing PPAR-γ.54 The study provides evidence that miR-34a decreases the mitochondrial content and increases TAG via PPARα and AMPK pathways by targeting the AdipoR2 gene.55 A number of factors regulate the transcriptional activation potential of C/EBP-β in stimulated preadipocytes. DNA binding of C/EBP-β is facilitated by MAPK phosphorylation beginning at 4 h post-stimulation and GSK3β phosphorylation-14 h into differentiation. MiR-34a regulates therapy resistance by targeting HDAC1.56 We have shown that the ability of C/EBP-β to activate C/EBP-β expression in preadipocytes stimulated to differentiate is initially reduced through the interaction of C/EBP-β with an mSin3A/histone deacetylase 1 (HDAC1) complex.57 PPAR-γ and C/EBP-β are marker genes of adipocyte differentiation. So, miR-34a can regulate adipocyte differentiation by targeting target genes.

MiR-126 Regulated Adipocyte Differentiation

MiR-126 is a single stranded small RNA molecule with a length of 23 nucleotides encoded by endogenous genes,58 which can widely mediate the regulation of physiological reactions such as cell differentiation, proliferation and migration.59 Functional analysis of miR-126 demonstrated that its overexpression conveys neurotoxicity by impairing IGF-1/PI3K/AKT signaling, and that its inhibition increases the trophic effects of IGF-1.60 Studies also confirmed that miR-126 exerted these pivotal functions by down-regulating the expression of CRK.61 During 3T3-Ll cell differentiation induction, C-CRK is phosphorylated on tyrosine by IGF-1 receptor kinase and dephosphorylated by PTPase.62 In addition, over-expression of miR126 down-regulated IRS-1 expression, suppressed AKT and ERK1/2 activation. Decreased expression of IRS-1 in embryonic fibroblast cells severely decreased the expression of C/EBPα and PPARγ.63 The inhibitory effect of mir-126 on VEFG expression was investigated and indicated that VEGF is a target of miR-126.64 Retrovirus-mediated restoration of VEGF expression in mutant cells reduced adipocyte differentiation to the levels exhibited by control cells.65 In a word, miR-126 played an important role in adipocyte differentiation.

MiR–143-3p Regulated Adipocyte Differentiation

miR-143 was identified to promote adipocyte differentiation by using antisense oligonucleotides.66 There are many target genes of miR-143-3p that play a regulatory role in adipocyte differentiation, such as MAPK7,67 MAP3K7,68 AKT,69 KLF5,70 PI3K71 and EZH2.72 Firstly, MAPK7 inhibited adipocyte differentiation73 and MAP3K7 induces adipocyte differentiation through PPARγ signaling.74 Secondly, AKT/PKB may play a role in suppression of apoptosis and negatively regulate preadipocyte differentiation.75 KLF5 is also induced by C/EBPβ/δ, and that it then acts in concert with C/EBPβ/δ to regulate PPARγ2 expression76 and EZH2-induced H3K27me3 of WNT gene promoters facilitated adipogenic differentiation of murine preadipocytes.77 Finally, IRSs/PI3K signal pathway may play an important role in the differentiation of 3T3-L1 preadipocytes by regulating the expression of C/EBPα and PPARγ.78 These results suggest that miR-143-3p can regulate adipocyte differentiation.

MiR–221/222 Regulated Adipocyte Differentiation

miR-221/222, located in a cluster on chromosome Xp11.3, are considered part of the same family. They share the same’seed’ sequence, short regions at their 5’ ends through which they bind their target sites in mRNA 3’-UTRs. Studies showed that miR-221 and 222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and activate the AKT pathway.79,80 SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-AKT-FOXO1-PPARγ pathway,81 which indicates PTEN, TIMP3 and AKT genes play an important role in adipogenesis. In addition, miR-221 and 222 inhibited the expression of p2782 and Genetic ablation of p27 in mice leads to adipocyte hyperplasia.83 In a word, miR–221/222 can regulate adipocyte differentiation by multiple pathways.

MiR-320 Regulated Adipocyte Differentiation

MiR-320 is involved in a variety of pathological processes, including cell proliferation and differentiation.114 The present results provided evidence that the miR-320/ELF3 axis regulated tumor progression via the PI3K/AKT signaling pathway.111 Activated form of PI3K, a critical target of IRS1 downstream, led to phosphorylation of phosphatidyl inositides and then activated the downstream main target AKT, which is pivotal in regulating 3T3-L1 preadipocyte differentiation.115,116 In addition, Data study indicates that miR-320 negatively regulates expression of ET-1, VEGF, and FN through ERK1/2.109 The adipocyte-specific transcription factor PPARγ can be phosphorylated by ERK1/2 to decrease its transcriptional activity and inhibit adipocyte differentiation.117 Finally, A luciferase assay confirmed that miR-320 binds to the 3’-untranslated regions of AdipoR1, which indicated AdipoR1 is a target gene of miR-320.112 CTRP6 regulates proliferation and differentiation of intramuscular and subcutaneous adipocytes through the AdipoR1 (Adiponectin Receptor 1)/MAPK pathway.118 So miR-320 can regulate adipocyte differentiation by targeting ERK1/2, PI3K and adipoR1.

METTL3 Regulated Adipocyte Differentiation by Directly Modifying Key Genes

Methyltransferase-like 3 (METTL3), a key RNA methyltransferase, has been demonstrated to regulate neurogenesis,119 spermatogenesis,120,121 early embryonic development,122 stem cell pluripotency in mice,122,123 and white fat cell differentiation in vitro.18 Recently, Yao et al found that METTL3 plays an important role in BMSCs differentiation and adipogenesis and there was a negative correlation between METTL3 expression and porcine BMSCs (pBMSCs) adipogenesis.124 It was demonstrated that the deletion of METTL3 significantly promoted the pBMSCs adipogenesis process and janus kinase 1 (JAK1) protein expression via an m6A-dependent way.124 Specifically, METTL3 inhibited pBMSCs adipogenic differentiation by targeting the JAK1/STAT5/C/EBPβ pathway via an m6A-YTHDF2–dependent manner.124 C/EBPβ is a marker gene of adipocyte differentiation, which indicates METTL3 plays an important role in regulating adipocyte differentiation.

Effect of Adipocyte Differentiation and on Fat Deposition

Fat deposition is the main means of energy storage in animals. Mammalian adipose tissue mainly exists in four forms: subcutaneous, visceral, intermuscular and intramuscular fat. Generally, the differentiation of adipocytes refers to the process of preadipocytes differentiating into multi compartment adipocytes.125,126 After 8 days of culture in vitro, precursor adipocytes were induced to differentiate into mature adipocytes by PPARγ, CEBP/a and FABP4.127 The number and volume of lipid droplets in mature adipocytes increased. At the same time, the volume of mature adipocytes also increased significantly, which also increased the content of adipose tissue. So, adipocyte differentiation promoted fat deposition.

Conclusions

In summary, although the correlation between m6A modification and fat deposition, as a hotspot in the field of genetics, has been extensively explored, most studies concentrated on gene sequencing analysis, differential expression analysis, and modification site analysis. There are few studies on the functional phenotypes and mechanisms of action at the cell level, but studies in this field are likely to be key to revealing the origin of fat deposition, especially the origin and development of obesity. With a deep understanding of mechanism of fat deposition and the targeted study for m6A modification, m6A modification then provides a new perspective for elucidating the occurrence and development of related obesity diseases, providing a new direction for guiding the diagnosis and treatment of obesity diseases.