Analysis of RNA binding proteins using stable cell lines

Abstract

Over 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.