Functional Study On Two Transcripts Of The TBC1D8B Gene In Human

The TBC domain proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulator of RABs. These proteins play key roles in cell signal transduction and diseases, especially in the development of tumor.Alternative polyadenylation (APA) has been gained attention because it occurs in approximately 70% eukaryotes genes. Most of genes possess more than one polyadenylation site (pA), leading to different transcript isoforms with various coding potentials and/or variable 3’untranslated regions (UTRs). Therefore, APA can also be regarded as a mechanism of new gene origin by using different cleavage sites. By a comparative genomics analysis, we found that the TBC1D8B gene in human may produce two transcripts (TBC1D8B-a and TBC1D8B-b) by APA. TBC1D8B-a is exist in almost all mammals, while TBC1D8B-b is a human-specific transcript. Although TBC1D8B is a member of the TBC domain protein family, the biology function of two transcripts (TBC1D8B-a and TBC1D8B-b) remains unclear.In this study, we first screened ACTB as the optimal reference gene for qPCRanalysis using several algorithms. Next, we carried out the functional studies on the TBC1D8B-a and TBC1D8B-b transcripts. The results showed:1) The 3’UTR sequences of TBC1D8B-b gene highly conserv among different populations or diseases, suggesting that it may have importantly biological function.2) The TBC1D8B-b gene and its encoding protein Tbc1d8b-b are human-specific. The emergence of TBC1D8B-b gene is a rapid evolutional event.3) The TBC1D8B gene widely expressed in normal human tissues or cells. The relative expression of TBC1D8B-a is significantly higher in prostatic cancer, colorectal cancer and lung cancer than the corresponding normal tissues.4) The TBC1D8B-a gene can significantly promote the cell proliferation and DNA synthesis, accelerate the cell cycle processing, and improve the ability of cell migration and invasion. It has the characteristics of proto-oncogenes.Interestingly, these functions would lost when the two curial amino acids in active sites of TBC domainwere mutated (Arg538Lys and Gln573Leu), indicating that the function of TBC1D8B-a depends on the activity of TBC domain.5) The wild TBC1D8B-b gene has no effect on promoting cell proliferation, accelerating cell cycle progression and improving the ability of cell migration. However, the TBC1D8B-b mutant (Arg538Lys and Gln573Leu) could gain the phenotypes.6) The possible signal pathways of the wild TBC1D8B-a and the TBC1D8B-b mutant were predicted using high throughput sequencing technology. The results showed that the two genes may be involved in different signaling pathways.This study not only enriched our understanding of TBC domain protein family, but also increased our understanding of the origin and functions about human-specific genes. The results may provide new targets for cancer diagnosis and drug treatment.