Figure 4

ASBEL forms duplexes with ANA/BTG3 mRNA and prevents its nuclear-cytoplasmic transportation.

(a) Schematic representation of the genomic organization of the region containing ASBEL and ANA/BTG3 and the ASBEL mutant constructs used in RIP assays. (b, c) RIP assay. Lysates prepared from JHOC5 cells transfected with the wild type, antisense (Antisense-ASBEL) or mutant pp7-ASBEL (ASBEL fused to a pp7 coat protein-binding sequence), ANA/BTG3 mRNA and FLAG-tagged pp7 coat protein expression constructs were subjected to immunoprecipitation with anti-FLAG antibody followed by RT-PCR analysis to detect ANA/BTG3 mRNA. Four mutants shown in (a) were used in (c). The apparent immunoprecipitation efficiency for a specific RNA was calculated by dividing the amount of RT-PCR product obtained in the immunoprecipitated sample by the amount obtained from the input RNA. HPRT1 was used as a negative control. (d) Subcellular distribution of ANA/BTG3 mRNA and ANA/BTG3 protein. JHOC5 cells transfected with siRNA targeting ASBEL or control siRNA were subjected to subcellular fractionation. (Left) The amounts of the indicated RNAs in each fraction were evaluated by RT-PCR analysis. U1 was used as a loading control. (Right) The cytoplasmic fraction was subjected to immunoblotting analysis with antibodies against the indicated proteins. α-tubulin was used as a loading control.