Molecular Mechanism Of The Regulation Of Adipogenesis By NSUN2 In 3T3-L1 Preadipocyte

The fat deposition of animals has been a key element for the growth performance and meat quality of husbandry for a longtime.The over-accumulation of adipose tissue reduced the efficiency in feed utilization and affected animal health and food safety.Less deposition of the adipose tissue leaded the lower meat quality.Therefore,identifing the regulatory mechanism of fat deposition in animals has became a focal of husbandry.Both hyperplasia and hypertrophy were key determining factors for increasing the number and the size of adipocytes during adipose tissue growth.Mitotic clonal expansion was a necessary stage during adipogenesis.In this stage,the cells which remained in the contact inhibition stage will reenter cell cycle progression,resulting in an increase numbers of cells by mitosis.Currently,a large number of studies revealed an association between RNA modifications and adipogenesis.It was mainly focusing on how m6A RNA methylation regulated the adipogenesis.But the regulation mechanism of adipocyte differentiation by m5C RNA methylation has been reported scarcely.NSUN2(NOP2/Sun RNA methyltransferase family member 2)was a methyltransferase of m5C modification in mammalian mRNAs.The previous studies indicated that NSUN2 was essential for the proliferation and differentiation of stem cells.Bescides,NSUN2 was reported as a cell cycle regulator.But the effect of NSUN2 on preadipocyte differentiation was still unknown.Therefore,the regulation mechanism of NSUN2 in adipogenesis was demonstrated in this study.Exp.Ⅰ Effect of NSUN2 on adipogenesisThe 3T3-L1 cells which knockdown or overexpression of NSUN2 has been induced 18 hours or 7 days to test the effect of adipogenesis.The results were as follows:1)NSUN2 negatively regulated lipid accumulation:The lipid accumulation and the expression of adipogenesis related genes were increased in NSUN2-knockdown cells(P<0.05)and impaired in NSUN2-overexpressed cells compared to control(P<0.05).2)NSUN2 negatively regulated cell cycle progression:Overexpressing NSUN2 resulted in a significant inhibition of G1 phase in cell cycle progression(P<0.01).These results indicated that NSUN2 negatively regulated cell cycle progression and adipogenesis.Exp.Ⅱ Effect of methyltransferase activity of NSUN2 on p21 protein expressionUsing NSUN2 knockdown and overexpressed cells to detect the protein levels of cell cycle related genes.Revealing the effect of NSUN2 on m5C modification of cell cycle related genes.The results were as follows:1)NSUN2 positively regulated p21 protein levels.It was significantly decreased in NSUN2 knockdown cells(P<0.01)and increased in NSUN2 overexpressed cells(P<0.05).2)NSUN2 regulated p21 expression depending on the activity of methyltransferase:Using NSUN2 methyltransferase activity mutant plasmid to detected the mechanism of p21 expression.The result indicated that NSUN2-mutant transfected cell line made no difference of its regulatory effect on p21 protein expression and lipid accumulation compared to control(P>0.05).3)NSUN2 regulated cell cycle progression and lipid accumulation of 3T3-L1 cells depending on the activity of methyltransferase:NSUN2 mutant plasmid inhibited the progression of cell cycle(P>0.05)and lipid accumulation of 3T3-L1 preadipocytes.4)NSUN2 enhanced the m5C level of p21 mRNA:Using MeRIP experiment to test whether NSUN2 methylated p21 mRNA.The NSUN2 wild type plasmid exhibited a remarkable increase of m5C content in p21 mRNA(P<0.01).The m5C content in p21 mRNA made no difference between NSUN2 mutant and control cells(P>0.05).These results indicated that methyltransferase activity of NSUN2 played an important role on p21 expression and adipogenesis.Exp.Ⅲ Effect of NSUN2 on p21 mRNA translation efficiencyUsing Polysome Fraction Analysis to detected the translation efficiency of p21 mRNA.Detecting the regulation mechanism of NSUN2.The results were as follows:1)NSUN2 positively regulated the translation efficiency of p21 mRNA:The p21 mRNA level was unchanged in NSUN2 overexpressed and depleted cells line compared to control(P>0.05).The result of polysome fraction analysis experiment indicated that the translation efficiency of p21 mRNA was decreased in NSUN2 depleted cells(P<0.01)and increased in NSUN2 overexpressed cells(P<0.01).2)The m5C reading protein,Alyref,positively regulated p21 expression:Losing Alyref will put out a decreased p21 expression in NSUN2 overexpressed cells.The p21 expression of Alyref depleted cells was reduced.3)Alyref bound to p21 mRNA and accelerated the export efficiency:The result of RNA immunoprecipitation experiment revealed that p21 interacted with Alyref protein depending on m5C manner.Alyref selectively recognized m5C modification and simultaneously achieved the nuclear-cytoplasmic shuttling depending on an m5C-dependent manner.The result of subcellular fraction and fluorescence in situ hybridization experiment showed that Alyref mediated p21 mRNA export to cytoplasm(P<0.01).These results demonstrated an m5C-dependent regulatory function of NSUN2 and Alyref in mRNA export during cell cycle progression of preadipocytes.In this study,we demonstrated an observed cell phenotypic change that NSUN2 inhibited adipogenesis by hindering cell cycle progression in early adipocyte differentiation.Further studies indicated that NSUN2 promoted the protein expression of a critical cell cycle regulator,p21.in an m5C dependent manner.These suggested a previously unstated relationship between NSUN2 and cell cycle progression during adipocyte differentiation.Further studies demonstrated that NSUN2 cooperated with Alyref to mediate p21 mRNA export and promoted the translation efficiency of p21 mRNA.Our work revealed a crucial function of NSUN2-m5C-Alyref signaling in adipogenesis.which provieded a new prospect of the effects of mRNA m5C methylation in fat deposition and meat quantity.