Expression Pattern, Developmental Regulation And Functions Of Pre-mRNA Splicing Protein Tra2 Genes In Neural Tissues

Tra2 (Transformer 2) is a family of pre-mRNA splicing proteins and expressed in mammalians as well as in drosophila. In drosophila, Tra2 is a key regulatory protein in sex differentiation. However, in mammalians, the mechanism of sex determination is different from that in drosophila. It indicates that Tra2 might have other functions in mammalians. Limited studies have implied that Tra2 proteins might be involved in mammalian development, but its expression pattern, developmental regulation and physiological functions and mechanism are still unknown. In this thesis, my study focused on the expression and function of Tra2 in mammalian development, especially in neural development.Analysis of gene expression pattern is an important aspect in the study of gene function. Up to date, several mRNA Tra2 isoforms (Tra2@1,2,3,4,5 and Tra2a) have been identified, however, their precise functions are unknown. We hypothesize that if Tra2 contributes to the regulation of development, then as an aspect of functional regulation, the expression patterns of its isoforms would be regulated depending on the developmental stages and the types of tissues. Following the hypothesis, in this thesis, we first analyzed the expression pattern of Tra2 isoforms at different developing stages in different tissues, including the detection of Tra2 new isoforms possibly expressed during the development. In the study we used the RT-PCR to amplify Tra2a isoforms in human fetal tissues and identified a new isoform (Tra2a2) that shares high similarity with the previously reported Tra2a (Tra2al). In addition, to analyze if there are differences in the expression amount of Tra2 isoforms at different developing stages or in different tissues, a semi-quantitative RT-PCR analysis was performed to characterize the expression pattern of Tra2 isoforms in tissues at different developing stages (11-week and 16-week), the results showed that the expression of Tra2@1, 5 and Tra2al varied depending on the developmental stages, but was relatively invariable between different tissues. The expression of Tra2@3 was more restricted depending on the development stages and type of tissues. There wasno detectable expression in all the tissues at 11 week while ubiquitous expression at 16 week, although the expression amount varied significantly in various tissues. For Tra2p4, the expression pattern was similar to that of Tra2p3, with restricted expression at 11 week in several tissues including the cerebellum and ubiquitous expression at 16 week. For Tra2a isoforms, Tra2al and Tra2A2, the expression was ubiquitous in various tissues at11 week and 16 week, with the increased expression of Tra2al in all tissues and Tra2a2 in most of tissues except the large intestine and spleen following the development from 11 week to 16 week. These results imply at transcript expression levels that during human development, including neural development, the need for Tra2 proteins in regulation of pre-mRNA splicing varies depending on tissue types and developing stages.Studies have shown that Tra2 proteins are functionally involved in regulation of pre-mRNA splicing. Therefore, compared with the study at mRNA levels, the study of Tra2 expression during human development at protein levels would more directly reflect the function of Tra2 protein in the development. Therefore, it is necessary to detect if there is any relation between the expression level of Tra2 proteins and human development. We firstly prepared the antibodies specific to Tra2 protein isoforms. By comparing the coding sequences of Tra2a and Tra2p isoforms, the peptide fragments specific to Tra2al and Tra2l were identified and expressed as GST-fusion proteins in bacteria. The purified Tra2 fusion proteins were used to immunize New Zealand rabbits to generate the antiserums specific to Tra2al and Tra2pl proteins. After determination of the immuno-specificities of the generated antibodies, the two kinds of antibodies (anti-Tra2al and Tra2pl antibodies) along with the other antibody (RA301 antibody) that is theoretically a...