Analysis of RNA binding proteins using stable cell lines

PhD ThesisOver 94% of human genes are alternatively spliced. Alternative splicing regulation is necessary for some developmental pathways. Many proteins including RNA binding proteins such as STAR family, hnRNPs, SR and SR-like proteins are involved in alternative splicing regulation. Some members of these proteins also have important roles in gene expression, transcription, signal transduction, RNA metabolism, cell cycle regulation and cancer. Although these proteins are known to be involved in alternative splicing control of specific targets, mechanisms of many of their actions and protein interaction partners are unknown. The aim of present study was the identification and characterization of protein interaction partners of some of these proteins including: STAR family (T-STAR, Sam68), hnRNP proteins (hnRNP G, hnRNP G-T) and SR-like proteins (Tra2β). The cDNAs of T-STAR, wild type Sam68 and P439R mutant Sam68 were cloned into a FLAG epitope encoding vector. The next step was generation of stable cell lines expressing these FLAG tagged proteins. To induce protein expression, stable cell lines were treated with tetracycline. The wild type Sam68-FLAG tagged protein was nuclear, while the P439R mutant Sam68-FLAG had cytoplasmic localization. Using immunoprecipitation and mass spectroscopy, both FLAG tagged proteins and co-purified proteins were purified and identified. RHA (RNA helicase A) was detected as an interacting partner for T-STAR, wild type and P439R mutant Sam68. Also, CCAR1 (cell division cycle and apoptosis regulator 1) was detected as a further partner for wild type and mutant Sam68. In addition, stable cell lines expressing FLAG tagged hnRNP G, hnRNP G-T and Tra2β were generated. These proteins and their candidate protein partners were pulled down and detected using immunoprecipitation and mass spectroscopy. Some of these detected proteins such as hnRNP C, hnRNP CL1 and RNA binding motif protein, X-Linked-Like 1 were common interacting candidates for both hnRNP G and hnRNP G-T. In addition to confirmation the roles of hnRNP G and hnRNP G-T in reduction of cell growth, the over-expression of hnRNP G-T led to remarkable cell morphological changes and alternative splicing of some target genes. In conclusion, in this project new interacting protein partners for T-STAR, Sam68, hnRNP G, hnRNP G-T were detected which their roles need to be tested within the cell

Expression of Tra2β in Cancer Cells as a Potential Contributory Factor to Neoplasia and Metastasis

The splicing regulator proteins SRSF1 (also known as ASF/SF2) and SRSF3 (also known as SRP20) belong to the SR family of proteins and can be upregulated in cancer. The SRSF1 gene itself is amplified in some cancer cells, and cancer-associated changes in the expression of MYC also increase SRSF1 gene expression. Increased concentrations of SRSF1 protein promote prooncogenic splicing patterns of a number of key regulators of cell growth. Here, we review the evidence that upregulation of the SR-related Tra2β protein might have a similar role in cancer cells. The TRA2B gene encoding Tra2β is amplified in particular tumours including those of the lung, ovary, cervix, stomach, head, and neck. Both TRA2B RNA and Tra2β protein levels are upregulated in breast, cervical, ovarian, and colon cancer, and Tra2β expression is associated with cancer cell survival. The TRA2B gene is a transcriptional target of the protooncogene ETS-1 which might cause higher levels of expression in some cancer cells which express this transcription factor. Known Tra2β splicing targets have important roles in cancer cells, where they affect metastasis, proliferation, and cell survival. Tra2β protein is also known to interact directly with the RBMY protein which is implicated in liver cancer