Fig. 5
ALKBH5 regulates Cdh2 mRNA translation via the IGF2BP1/2/3 complex and basal endoplasmic specialization via N-cadherin. a Polysome profiling analysis for detecting the translation efficiency of Cdh2 mRNA in TM4 cells with and without Alkbh5 knockdown. Western blot analysis was performed by using RPS6 and RPL11 to detect the efficiency of polysome fractionation. b RIP–qPCR to detect the interaction between IGF2BP1, IGF2BP2, or IGF2BP3 proteins and Cdh2 mRNA. c Coimmunoprecipitation to detect the interaction between IGF2BP1, IGF2BP2, and IGF2BP3 proteins. d Western blot analysis to detect the expression of IGF2BP1, IGF2BP2, and IGF2BP3 after silencing Alkbh5 in TM4 cells. β-Actin and GAPDH were used as internal controls. e Western blot analysis to detect the expression of N-cadherin after knocking down Igf2bp1, Igf2bp2, or Igf2bp3. β-Actin was used as an internal control. f Western blot analysis showing the expression of N-cadherin before and after knocking down Alkbh5. GAPDH was used as an internal control. g Some key BTB-related molecules detected by western blot analysis. β-Actin was used as an internal control. h Images showing the ultrastructure of basal endoplasmic specialization (marked by red arrow) in the testis of WT or Alkbh5-KO mice as observed under transmission electron microscopy. Yellow arrow indicates actin bundles. i Schematic diagram elucidating the mechanism of ALKBH5 in regulating the integrity of the blood–testis barrier. Under physiological conditions with normal ALKBH5 expression, Cdh2 mRNA is maintained at a relatively low m6A level and is kept away from the IGF2BP1/2/3 complex and YTHDF1-mediated translation of Cdh2. Thus, N-cadherin is expressed at a reasonable level to maintain the stability of basal endoplasmic specialization and integrity of the blood–testis barrier. However, insufficient ALKBH5 protein expression leads to hypermethylation of Cdh2 mRNA and further facilitates its translation under the action of the IGF2BP1/2/3 complex and YTHDF1. Consequently, overexpression of N-cadherin disrupts the stability of basal endoplasmic specialization and integrity of the blood–testis barrier. *P < 0.05, **P < 0.01, ***P < 0.001