碩士 / 中山醫學大學 / 生物醫學科學學系碩士班 / 99 / CUG-binding protein 2 (CUGBP2) is a member of the CUG-binding and ETR-3 like factors (CELF) protein family, and functions as mRNA alternative splicing regulator. Previous studies have shown that CUGBP2 is a trans-acting factor associated with human diseases, such that its mis-regulation results in aberrant splicing of various genes. Due to the alternative splicing regulation activity of CUGBP2, its function is also very important in fetal development. Orthologous proteins of CUGBP2 were found in difference species, and through homologous protein sequence alignment of CUGBP2 orthologs between human, mouse and zebrafish, a 98% similarity is observed in the RNA-recognition-motif region. Based on the above information, there presents high possibility that CUGBP2 and its orthologs in mouse and zebrafish are able to recognize similar motif pattern within the species. Here, we used bioinformatics aids to predict candidate genes whose alternative splicing is regulated by CUGBP2.
The bioinformatics approaches are based on three major strategies. First is the CUGBP2 recognition pattern from the sequence consensus analysis of known CUGBP2 splicing-regulated genes, that is, the recognition of CUGBP2 binding motifs within the flanking regions of an alternatively spliced exon. The second approach is supportive evidences in alternative splicing of genes in human, mouse and zebrafish to deternime alternative splice type of each exon. Lastly, through evidences in homology between genes in three species, we identified homologous exons that are potentially regulated by CUGBP2. Through these approaches we identified 60 candidate genes possibly regulated by CUGBP2, of which we chose 19 genes and tested their alternative splicing ratio changes under cugbp2 knockdown model in zebrafish embryonic development. Functional analysis showed that the 19 candidate genes are related to five major fields: brain/muscle related, transcription related, cell cycle/ differentiation/apoptosis related, possess enzyme activity and transport related. We used morpholino (MO) microinjection knockdown system to knockdown cugbp2 in zebrafish. MO knockdown caused abnormal phenotypes in zebrafish embryos and developmental delay. We then used PCR semi-quantification method and tested the isoform splicing ratio changes of candidate genes. Our results showed 3 candidate genes with isoform ratio changes, two of which is showed an indirect regulation by cugbp2 (tpd52l2 and neo1 gene). The other gene, col4a3bp gene, is known for its regulation in zebrafish muscle and brain tissue development, showed ratio changes during somite development stages in zebrafish embryos.
| Identifer | oai:union.ndltd.org:TW/099CSMU5114006 |
| Date | January 2011 |
| Creators | Shu-Yu, 劉書瑜 |
| Contributors | 劉玉凡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 109 |
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