Effects And Mechanisms Of Kev MRNA M~6A Genes In Porcine Intramuscular Fat Deposition

Intramuscular fat(IMF)content has been generally recognized as a desirable trait in pork meat because of its positive effect on eating quality.The Jinhua pig,one of Chinese excellent local pig breeds with outstanding pork quality,shows an intramuscular fat content exceed 3%.In contrast,the Landrace pig,a Danish breed known for having a lean carcass with plain meat quality,whose intramuscular fat content is relatively low.Therefore,Jinhua pig and Landrace pig can be used as ideal experimental animal models for the study of intramuscular fat.N6-methyladenosine(m-1)is the most prevalent chemical modification in eukaryotic mRNA,and previous studies reported that m6A modification downregulates adipogenesis in porcine adipocytes,and it may affect adipogenesis by regulating alternative RNA splicing.However,whether m6A modification is a driving force behind adipogenesis in intramuscular preadipocytes remains largely unknown.To better understand the molecular mechanisms underlying the miA contribution to fat deposition in muscle,we used the longissimus dorsi muscles(LDM)of Jinhua pigs and Landrace pigs for comparative analyses of whole transcriptome-wide m6H profiling,the differential gene of m-A modification of these two muscles were analyzed.And the underlying molecular mechanisms were conducted on porcine intramuscular preadipocytes.Results are as bellow:Exp.? Comparative study of intramuscular fat deposition and mRNA m6A levels in Jinhua and Landrace pigsThe 180-day-old obese-type Jinhua and lean-type Landrace pigs were used as models to compare the differences in intramuscular fat deposition between the two pigs.The intramuscular fat content of 180-day-old Jinhua pigs was significantly higher than that of Landrace pigs(p<0.05).The results of muscle tissue section staining showed that the size and number of intramuscular fat cells in Jinhua pigs were higher than those in Landrace pigs.The m6A/A ratio of the LDM and psoas major muscles(PMM)from the two pig breeds was compared using high-performance liquid chromatography coupled with a triple quadruple tandem mass analyzer.The ratio was higher in Landrace(L-LDM)than in Jinhua(J-LDM),which varied from 0.6 to 0.9%.On the other hand,the expression levels of demethylase FTO were lower in Landrace than in Jinhua,whereas the levels of methyltransferase METTL3 showed the opposite trend.The results suggested that m6A of mRNA in LDM is negatively associated with fat deposition in skeletal muscle.Exp.? m6A-seq and analysis of skeletal muscle transcriptome in Jinhua pig and Landrace pigThe MeRIP-seq analysis of L-LDM and J-LDM yielded high-confidence m6A peaks within thousands of coding gene transcripts.We detected 3301 m6A transcripts in L-LDM and 2978 m6A transcripts in J-LDM samples.The m6A modification in pig LDM were highly consistent with patterns identified in other mammals,which shared the characteristics that:(1)m6A peaks are concentrated in the CDS and 3TUTR of each mRNA,especially at the junction of 3'UTR and CDS;(2)m6A modification tends to occur on the RNA motif RRACH sequence(where R = A/G,A = m6A,and H= A/C/G);(3)About 1/3 transcripts were modified with m6A,and about 1.7 m6A peaks per transcript.To predict the functions associated with m6A-modified genes,we conducted a gene ontology(GO)enrichment analysis,results showed that the common m6A modification gene of the two breeds were mainly involved in functional processes such as gene expression,muscle differentiation and development;the unique m6A genes(UMGs)of J-LDM are involved in intracellular protein metabolism,carbohydrate metabolism,mitochondrial composition,etc.While the UMGs of L-LDM are involved in skeletal muscle development and vascular development,etc.Most of lipid metabolism-related genes were involved in oxidation-reduction and lipid oxidation,102 unique m6A modified genes in J-LDM were riched in oxidation,while no oxidation related genes were unique to L-LDM.The results suggested that m6A modification of oxidative metabolism genes may regulate intramuscular fat deposition.Exp.? m6A enhances MTCH2 protein expression via YTHDF1 and promotes adipogenesisAmong the unique m6A genes associated with oxidation in J-LDM,we selected MTCH2,which exhibited significantly higher m6A modification in J-LDM than in L-LDM.We isolated and cultured porcine intramuscular preadipocytes to investigate the function of MTCH2 gens in regulating intramuscular fat deposition using RNAi and gene overexpression technology.Short interfering RNA-mediated MTCH2 knockdown of intramuscular preadipocytes decreased lipid accumulation.However,enforced MTCH2-expression in intramuscular preadipocytes significantly enhanced lipid accumulation,indicating that MTCH2 promoted adipogenesis of intramuscular preadipocytes.Furthermore,we investigated the effects of m6A modification of MTCH2 on adipogenesis in intramuscular preadipocytes.First,we searched for m6A modification sites in MTCH2,and a search of the sequence corresponding to the conserved m6A motif,RRACH,revealed a putative m6A site in the first 120 nucleotides of MTCH2 located at position 8 of the CDS(ATGGCGGACGCGGCCAG).Here,we introduced synonymous mutation at the putative m6A site on CDS(MTCH2-MUT),and then evaluated the m6A level of MTCH2-WT and MTCH2-MUT using MeRIP-qPCR.As expected,the m6A level decreased in MTCH2-MUT as compared with that of MTCH2-WT(P<0.01),indicating that the predicted site was modified by m6A.Intramuscular preadipocytes were transfected with MTCH2-WT and MTCH2-MUT,and the results indicated that MTCH2-MUT markedly decreased adipogenesis as compared with that of MTCH2-WT(P<0.05).We evaluated protein levels during adipogenesis and found that MTCH2 protein expression was higher in the MTCH2-WT than in MTCH2-MUT(P<0.05),thus explaining the greater lipid accumulation in the former.Overexpression of FTO suppressed the expression of MTCH2 in the first 2 days after MDI induction(P<0.05),suggesting that m6A modification may increase MTCH2 expression in adipogenesis.MTCH2 mRNA stability experiment reveled that there was no difference in the half-life of mRNA between MTCH2-WT and MTCH2-MUT groups,suggesting that YTHDF2 does not modulate MTCH2 mRNA stability.To determine whether YTHDF1 regulates MTCH2 expression,we introduced siYTHDF1 to knockdown YTHDF1 in both MTCH2-WT and MUT,the results showed a significant decrease in MTCH2 expression after YTHDFI knockdown in MTCH2-WT(P<0.05),as well as the Oil red O staining,whereas no significant change could be found in the MUT group.Overexpression of YTHDF1 and induction of adipogenesis resulted in an increase in MTCH2 protein expression in the first day of adipogenic differentiation,suggesting that YTHDF1 regulates MTCH2 expression during adipogenesis in intramuscular preadipocytes.To confirm whether YTHDFl functions as a direct m6A reader of MTCH2,we carried out RIP-qPCR experiments with lysates of porcine intramuscular preadipocytes overexpressing YTHDF1.Precipitation with the anti-FLAG antibody enriched MTCH2 mRNA as compared to the IgG control suggested that YTHDF1 directly binds to the MTCH2 mRNA,thus partly explaining the higher protein expression detected in MTCH2-WT.Together,these findings suggest that m6A modification of porcine MTCH2 promotes adipogenesis by enhancing YTHDF1-dependent mRNA translation.In summary,this study was the first to uncover the function of m6A modification in intramuscular fat deposition.Furthermore,the direct connection between MTCH2 expression and m6A status in the context of intramuscular preadipocytes adipogenesis sheds new light on our understanding of the biological significance of RNA m6A modification.