利用生物資訊方法預測cugbp2蛋白在斑馬魚胚胎發育時期所會調控其選擇性剪輯之下游基因

CUG-binding protein 2 (CUGBP2) 是CUG-binding protein and Embryonic lethal abnormal vision type RNA-binding protein-3 Like Factors (CELF) 蛋白質家族的成員之一。該蛋白質主要的功能為調控生物體中選擇性剪輯的機制。先前的研究指出CUGBP2是一個選擇性剪輯的trans-acting 因子，當失調時會造成人類疾病，如: 肌強直症 (myotonic dystrophy)，並且CUGBP2的選擇性剪輯調控，在生物發育過程中扮演重要的角色。CUGBP2的同源基因在很多物種中都有找到，其中在人類、小鼠及斑馬魚中CUGBP2蛋白序列的RNA recognition motif (RRMs) 相似度高達98%。因而推測在這些模型生物中的RRMs所辨識的RNA motif pattern相類似。因此利用了生物資訊學的方法來預測在人類、小鼠及斑馬魚中CUGBP2所會調控選擇性剪輯之基因。主要的方法1) 利用已知的文獻資訊中已知的實驗結果，推論出CUGBP2的結合序列，2) 並利用資料庫中的三個物種中Transcripts，推算出外顯子的選擇性剪輯資料，以及3) 整合同源性的外顯子來提高預測可信度。我們從結果中做了基因的評分並預測出60個候選基因，其中選擇19個候選基因，在斑馬魚中利用RNA顯微注射的實驗來證實。本實驗利用morpholino (MO) 顯微注射系統，將斑馬魚胚胎中的cugbp2 程度性的knock-down，來觀察胚胎生長遲緩並且發生畸形的現象。而藉由RT-PCR的實驗半定量的方法，來觀察cugbp2 knock-down之後候選基因之不同選擇性剪輯的isoforms的比例發生改變。目前根據結果證實有3個基因的選擇性剪輯的調控受到影響，其中兩個基因 (tpd52l2和neo1) 的剪輯改變是屬於間接性的調控，而另一個基因，col4a3bp基因，則是在cugbp2 knock-down後，魚體節發育時期isoform A和B之間表現量有比例上的改變。本篇論文利用生物資訊學的方法，預測了60個會有可能受到cugbp2調控的候選基因，並且在斑馬魚胚胎的cugbp2 knock-down實驗中證實了有3個候選基因確實受到影響。

關鍵字

斑馬魚胚胎發育；選擇性剪輯

並列摘要

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.

並列關鍵字

alternative splicing ； zebrafish embronic development

參考文獻

Barreau, C., Paillard, L., Mereau, A., and Osborne, H.B. (2006). Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions. Biochimie 88, 515-525.

Barron, V.A., Zhu, H., Hinman, M.N., Ladd, A.N., and Lou, H. The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation. Nucleic Acids Res 38, 253-264.

Black, D.L. (2000). Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367-370.

Brimacombe, K.R., and Ladd, A.N. (2007). Cloning and embryonic expression patterns of the chicken CELF family. Dev Dyn 236, 2216-2224.

Castle, J.C., Zhang, C., Shah, J.K., Kulkarni, A.V., Kalsotra, A., Cooper, T.A., and Johnson, J.M. (2008). Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines. Nat Genet 40, 1416-1425.

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利用生物資訊方法預測cugbp2蛋白在斑馬魚胚胎發育時期所會調控其選擇性剪輯之下游基因

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