The T-box proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. It is highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report post-transcriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla Luciferase reporter significantly reduced the activity of the Luciferase. Whereas deleting only the degradation elements from the 3'UTR resulted in reduced ac... More
The T-box proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. It is highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report post-transcriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla Luciferase reporter significantly reduced the activity of the Luciferase. Whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid mRNA between the two transgenes, with and without the 3'UTR, indicating the absence of post-transcriptional regulation of mid in MP2. Moreover, while elevated mid RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Over-expression of the two transgenes resulted in MP2-lineage defects about the same frequency. These results indicate a translational/post-translational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid RNA and the protein and had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the two transgenes.
