Many biological processes are controlled by mRNA expression, which is tightly regulated by a finely controlled genetic program. Alternative splicing of pre-mRNAs is an important mechanism for regulating gene expression in higher eukaryotes. Many genes implicated in DNA repair and apoptosis are expressed in response to ionizing radiation and most of these genes produce multiple variants of their mRNA.
When quantifying mRNA expression levels it is important to include in the analysis the mRNA from a control gene that is unresponsive to radiation. Housekeeping (Hk) genes are used as internal controls to normalize gene expression in real-time PCR. We examined the expression of Hk genes in four normal human lymphoblastoid cell lines and in one T-cell leukemia (Jurkat) cell line following exposure to different doses of ionizing radiation or various concentrations of phytohemagglutinin and phorbol myristate acetate. Our study showed that 18S rRNA and Beta- 2-microglobulin (B2M) appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells.
The BAX (BCl2 associated X protein) gene is known to respond to ionizing radiation and is a pro-apoptotic inducer of cell death. Bax mRNA has at least five splice variants (alpha, beta, delta, epsilon, and sigma). How these splice variants precisely regulate apoptosis is unclear. We quantified the mRNA expression levels of these five variants following radiation in normal human lymphoblastoid cells. BAX alpha, beta, sigma, and delta showed elevated expression following radiation. However, the BAX epsilon variant behaved very differently from the others in that there was a decrease in expression following radiation. This is the first study describing the expression of BAX splice variants following radiation.
In the process of cloning the alpha and epsilon variants to characterize them using a tetracycline expression system, we discovered new variants of Bax alpha and epsilon, each of which has an 87 base insert in between exons one and two. This insertion leads to a truncated protein containing 29 amino acids that appears to lack a functional domain. Over-expression of Bax alpha mRNA elevated the expression of caspases 3 and 7, confirming the role of Bax alpha as an inducer of apoptosis. In contrast, over-expression of the alpha+87 variant decreased caspase 3 and 7 expression. Over-expression of the Bax epsilon and epsilon+87 variants also resulted in a decrease in caspase 3 and 7 levels indicating that these epsilon variants also repress apoptosis. Thus, we are the first to show that some Bax variants are able to repress rather than induce apoptosis. This study attributes different functional roles to some of the BAX mRNAs, and also indicates that alternative splicing can act as a switch in controlling BAX mRNA expression to induce or repress cells to undergo apoptosis.
