Multiple alternative splicing forms of human RAD17 and their differential response to ionizing radiation

In this study, we have identified four alternatively spliced RAD17 RNAs, FM1, FM2, FM3, and FM4, which are produced through alternative splicing within the first 300 base-pairs of the coding region. FM3 and FM4 are two novel forms that have not been reported before. All four alternatively spliced RAD17 RNAs were detected in the tissues we exantined. However, the levels of these forms varied from tissue to tissue. The expression of these four forms was also found to differ in different phases of the cell cycle and following exposure to X-irradiation. FM2, FM1, FM4, and FM3 encode putative polypeptides consisting of 681, 670, 596, and 516 amino acids, respectively, To determine if these polypeptides were expressed in cells, we generated a polyclonal antibody using a synthetic peptide. A major band around 71 kDa and two minor bands around 73 and 62 kDa were detected in human normal fibroblasts on Western blots. These three bands appear to represent the proteins encoded by FM2 (the 73 kDa band), FM1 (the 71 kDa band), and FM4 (the 62 kDa band) since the apparent molecular weights are close to their theoretical weights of the predicted amino acid sequences. The abundance of the 71 kDa. protein was not significantly affected by X-irradiation, while the abundance of the 73 and the 62 kDa proteins was increased at least 5-fold 14 h postirradiation. The differential expression of these four alternatively spliced forms in different tissues, in different phases of the cell cycle, and their differential response to X-irradiation suggest that they may perform different functions in cell-cycle regulation and in the response to irradiation. (C) 2001 Elsevier Science B.V. All rights reserved.