Fig. 9
WTAP promoted TIMP4 mRNA degradation in a m6A YTHDF2-dependent manner. A The m6A modification sites on TIMP4 mRNA sequence were predicted by SRAMP, and found that there were 3 greater than moderate confidence m6A sites in TIMP4, including one high confidence m6A site and two moderate m6A methylation sites. B Chondrocyte was infected with shWTAP, shNC, mock, or WTAP, and then the m6A levels of TIMP4 was evaluated using MeRIP coupled with qRT-PCR. C The wild-type or mutant luciferase plasmids of TIMP4 m6A were cloned in pmirGLO. The mutant ones obtained A-G mutations on m6A motifs. D The luciferase reporter assay was used to detect the relative activity of the TIMP4- m6A-WT or TIMP4- m6A-Mut luciferase reporters in WTAP-knockdown SW1353 cells. E The mRNA expression of YTHDF2 in chondrocyte with or without LPS was detected by qRT-PCR. F The mRNA expression of YTHDF2 in mice OA cartilage tissue and mice normal cartilage tissue was detected by qRT-PCR. G The mRNA expression of YTHDF2 and TIMP4 was detected by qRT-PCR in chondrocyte infected with WTAP overexpressing or knockdown adenovirus. H RNA immunoprecipitation (anti-YTHDF2) in mock or WTAP-overexpression cells was conducted followed by RT-qPCR to measure the amount of TIMP4 mRNA binding to YTHDF2. I Chondrocyte was infected with WTAP overexpression adenovirus alone or in combination with YTHDF2 knockdown adenovirus, the mRNA expression of TIMP4 was determined by qRT-PCR. J Chondrocyte were infected with WTAP overexpression adenovirus alone or in combination with YTHDF2 knockdown adenovirus, the RNA decay rate was measured in chondrocyte treated with Actinomycin D. shWTAP, WTAP knockdown adenovirus; WTAP, WTAP overexpression adenovirus; shYTHDF2, YTHDF2 knockdown adenovirus; YTHDF2, YTHDF2 overexpression adenovirus. N = 3 ~ 6. n.sP < 0.01, **P < 0.01, and.***P < 0.001