The Mechanism Of STAU1 Promotes Adipogenesis By Regulating The Alternative Splicing Of Pparγ2 Pre-mRNA

Objective:To investigate how double stranded RNA binding protein STAU1 regulates alternative splicing of Pparγ2 m RNA to promote adipocyte differentiation.Methods:（1）STAU1 was knocked down and differentiation of 3T3-L1 preadipocytes was induced by cocktail method.The expression levels of Stau1,Pparγand Pparγ2 on the 4th day of differentiation were detected by RT-q PCR and Western Blot.The formation of lipid droplets on the 4th day of differentiation was observed by BODIPY staining.（2）The expression levels of Stau1,Pparγand Pparγ2 on the 4th day of differentiation were detected by RT-q PCR and Western Blot.The formation of lipid droplets on the 4th day of differentiation was observed by BODIPY staining.（3）3T3-L1 preadipocytes with STAU1 knockdown and differentiation on the 4th day were selected for RNA-seq and the expression changes after STAU1 knockdown were analyzed.（4）The frequency of different variable splicing was analyzed according to RNA-seq data,and the Minigene splicing reporter plasmid was constructed to analyze the effect of STAU1 on the variable splicing of Pparγ2 m RNA.（5）adipose tissue specific knockout STAU1 mice were constructed and fed normal diet and 60%high fat diet for 17 weeks,respectively,to detect body weight,lipid,GTT,ITT and other metabolic phenotypes,i WAT tissue size,adipocyte area and PPARγprotein expression.（6）SVF cells in i WAT of STAU1^fl/fl and STAU1^Δ/adipo mice were isolated and cultured,and induced to differentiate until day 10,respectively.The expression levels of Stau1,Pparγand Pparγ2 were detected at day 0,2,4,6,8,and 10.STAU1 was overexpressed by lentivirus in two groups of SVF cells,and the expression levels of STAU1,Pparγand Pparγ2 were detected on day 4 of induced differentiation.Results:（1）Compared with the control group,the generation of lipid droplets and the expression of Pparγ2 were significantly decreased by knockout of STAU1 during the differentiation of 3T3-L1,with statistical significance（P<0.05）.（2）RNA-seq results showed that there was a significant difference between the STAU1 knockdown group and the control group,and there was a significant correlation between biological repeats.Compared with the control group,1719 genes were up-regulated and 1464 genes were down-regulated in si RNA1 group,2336 genes were up-regulated and2718 genes were down-regulated in si RNA2 group,895 genes were up-regulated and 554genes were down-regulated in both groups.GESA enrichment analysis showed that PPARγsignaling pathway related gene expression was down-regulated by STAU1 knockdown,and the difference was statistically significant（P<0.05）.（3）RNA-seq results showed that knockdown STAU1 had the greatest effect on exon jumping events.Insertion of B1 in Minigene reporter plasmid significantly increased the splicing of downstream exons,with statistical significance（P<0.05）,while truncated insertion sequence significantly reduced the inclusion of downstream exons.The difference was statistically significant（P<0.05）.（4）Compared with STAU1^fl/fl mice,the expression of STAU1 in i WAT of STAU1^Δ/adipo mice was significantly decreased,but the expression of STAU1 in heart,liver,spleen,lung,kidney and spleen was not different.In HFD feeding condition,The body weight of STAU1^Δ/adipo mice was significantly lower than that of STAU1^fl/fl mice,and the serum triglyceride was significantly decreased,with statistical significance（P<0.05）.The results of GTT and ITT showed that STAU1^Δ/adipo mice had better glucose tolerance and insulin sensitivity,and the difference was statistically significant（P<0.05）.Compared with STAU1^fl/fl mice,the i WAT volume and adipocyte area of STAU1^Δ/adipo mice were significantly decreased,and the expression level of PPARγwas significantly decreased.（5）Compared with STAU1^fl/fl group,the expressions of Stau1 and Pparγin STAU1^Δ/adipo group during SVF differentiation were significantly decreased,with statistical significance（P<0.05）.After overexpression of Stau1 with lentivirus,Pparγexpression can be compensated.Conclusion:STAU1 promotes splicing of E1 exon by binding with the B1 sequence of Pparγ2 pre-m RNA,thus enhancing the expression of Pparγ2 and promoting adipocyte differentiation.