Background: RNA binding motif (RBM) proteins, RBM10v1, RBM10v2 and RBM5 share a high degree of the conserved domains. So far, the drug-sensitivities of the RBMs in tumor cells have not been fully examined. Objective: The expression profiles of RBM10 and RBM5 in several virus-transformed tumor cells, and the effect of the most established antitumor agents, actinomycin D and doxorubicin, were investigated. Methods and Results: Doxorubicin and actinomycin D differentially reduced RBM10 and RBM5 protein, respectively in both of HeLa and COS-7 cells. RBM10 protein was highly sensitive to doxorubicin in HeLa, COS-7 and A549 cells. In silico analysis revealed the several sumoylation sites of RBM10 and its sumoylated form could be targeted for the activated ubiquitin proteasome system. Actinomycin D affected the nuclear speckles localization of RBM10 and RBM5 in COS-7 and A549 lung carcinoma cells. Addition of actinomycin D in the culture medium and following culture for 3-4 hours promoted the prominent nuclear speckles of RBM10v2-GFP and RBM5.Hence, we explored the subnuclear localization of the full length RBM10v2 (852aa) and the amino terminally truncated forms and the responsible structural elements. The amino terminally truncated RBM10v2 [#486-852, #642-852], RBM10v2 [#648-852], RBM10v2 [#681-759, #681-852], and RBM10v2 [#660-852] retained the targeting elements for the nuclear speckles, nucleoplasm, nucleoli and whole nuclei, respectively. Conclusion: RBM10 is highly sensitive to doxorubicin. Actinomycin D affects the structural elements of RBM10 and promotes the nuclear speckles targeting. The C-terminal regions: RBM10v2 [#642-647], [ #642-659], and [ #660-680] play critical roles in the targeting to the subnuclear compartments.SIM and sumoylation sits of RBM10 and PML4 are important for molecular interaction of RBM10 and PML.HK-2 cells or human normal fibroblasts TIG3S. These cells were subdivided into three groups according to the RBM protein levels. Interestingly, HeLa and COS-7 cells were regarded as high RBM-expressing, and A549 and TIG3S cells were regarded as low RBM-expressing, whereas HK-2 cells displayed the lowest expression profiles.The antitumor agent sensitivities of RBM proteins were explored in HeLa, COS-7 and A549 cells. At first, the reductions of RBM10 and RBM5 proteins were differentially triggered by DOX and AcD, respectively. The proteolytic sensitivity of RBM10 following from the activation of ubiquitin-proteasome system will be discussed. AcD significantly promoted the targeting of RBM10 and RBM5 to the enlarged nuclear speckles. Finally, the novel structural elements which target RBM10 to the nuclear speckles, nucleolar or PML-nuclear bodies are proposed.
Materials and MethodsCell lines and cell culture: COS-7 and HeLa PS (3) cells were provided from JCRB (Japanease Cancer Research Resources Bank, Tokyo, Japan). COS-7 cells represent SV40-large T antigen transformed immortal cell line. A549 human lung cancer cells were purchased from ATTC. HK-2, a cell line of human kidney proximal tubule ...
