Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3′ UTR of BCL2 mRNA

RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2 β (Tra2 β ) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2 β antibody and microarray analysis identified a subset of Tra2 β -associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2 β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2 β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3′ UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3′ UTR different from BCL2α 3′ UTR. RIP assays with anti-Tra2 β and anti-Argonaute 2 antibodies, respectively, showed that Tra2 β bound to BCL2α 3′ UTR, and that Tra2 β knockdown facilitated association of miR-204 with BCL2α 3′ UTR. The consensus sequence (GAA) for Tra2 β -binding lies within the miR-204-binding site of BCL2 3′ UTR. Mutation of the consensus sequence canceled the binding of Tra2 β to BCL2 3′ UTR without disrupting miR-204-binding to BCL2 3′ UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2 β -silenced or overexpressed cells revealed that Tra2 β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2 β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2 β regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.