系統識別號 U0026-0812200913560474
論文名稱(中文) DBY基因在體細胞之後轉錄機轉研究
論文名稱(英文) A study on post-transcriptional regulation of DBY, a germ-cell specific sterile gene, in somatic cells
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 95
學期 2
出版年 96
研究生(中文) 林育如
研究生(英文) Yu-ru Lin
電子信箱 t1694404@mail.ncku.edu.tw
學號 t1694404
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 口試委員-黃怡萱
中文關鍵字 DBY 基因  後轉錄調控機制  男性不孕症 
英文關鍵字 male infertility  post-transcriptional regulation  DBY gene 
中文摘要 大約10~15%夫婦受到不孕症的困擾,而其中有一半是因為男性不孕而導致的,約有10%造精功能障礙的不孕男性可以檢測到Y染色體的顯微刪除。DBY位在Y 染色體長臂上的AZFa區域,在維持早期精子細胞發育的過程中扮演重要的角色,因此DBY基因缺失會造成嚴重造精功能缺陷。DBY由660個胺基酸所組成,屬於DEAD box 蛋白家族的成員之一,含有RNA helicase motif以及高度保留的DEAD box motif。在之前的研究指出,DBY經選擇性聚腺苷酸化產生兩種不同的訊息RNA,其中一個全長2319個鹼基只表現在睪丸組織中,另外一個全長4416鹼基為廣泛組織表現,兩者的差異只在於3’端非轉譯區域。另外,有研究指出DBY的蛋白質只會在精祖母細胞到細線期精母細胞的階段被合成,因此,認為某些轉錄後調控機制會影響DBY蛋白質的合成。
這個實驗主要的目的是找出在DBY非轉譯區域中具有調控功能的序列以及相互作用的蛋白質。我們利用5端快速放大基因末端的方法找出在人類睪丸組織裡面,DBY有四種不同長度的5端非轉譯區域,其中69以及104鹼基廣泛表現在人類組織中,309和386鹼基則是只會在睪丸組織表現。DBY有三種不同長度的3端非轉譯區域,我們的實驗發現最長的3端非轉譯區域廣泛表現在睪丸以外的組織中,進一步的生物資訊分析發現,這段區域含有對於調控RNA穩定性及轉譯能力相當重要的第一型AU-rich element (Cordin et al.)。利用RNA-EMSA, UV-crosslink, and biotin pull-down等實驗技術,我們找到一個RNA結合蛋白 (HuR) 會和3端非轉譯區域的第一型ARE作用,接著在細胞裡大量表現HuR,利用冷光報導分析系統研究HuR與3端非轉譯區域的第一型ARE的交互作用,對於DBY轉譯調控的影響。在細胞裡面大量表現HuR會抑制DBY蛋白質的合成,但不影響其訊息RNA的穩定性。
英文摘要 About 10-15% health couples are infertile, and half of these cases could be attributed to a male factor. A significant proportion of infertile males are affected by spermatogenic defect. In cases with severe spermatogenic defect, approximately 10% of cases have microdeletions of the Y chromosome. The Y chromosomal azoospermia factor (AZF) is essential for human spermatogenesis. It has been mapped to Yq11 by deletion mapping analysis to three subintervals, which are named as AZFa, AZFb and AZFc. DBY has been reported as an AZFa candidate, of which deletion results severe spermatogenic defect. DBY belongs to DEAD box protein family, which are putative RNA helicases characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD). In previous studies, DBY was shown to have a ubiquitous transcript and at least one testis-specific transcript. These transcripts share the same protein cording region, but differ in 5’ and 3’untranslated region (UTR). Another study reported that translation of DBY is ubiquitously repressed except for spermatogonia and leptotene spermatocytes.
This study was conducted to test the hypothesis that cis-regulatory element is essential for translational control of DBY. We also tried to identify trans-acting factors which may be involved in pos-transcriptional control of DBY. Using RACE, we identified four different length of 5’UTRs in human testis: two of them are transcribed ubiquitously, one is 69 bp and the other is 104 bp long, while two longer transcripts, 309 bp and 386 bp in length respectively, are only expressed in testis. There are three types of 3’UTR in the database and the long form is abundant in various human tissues except testis. From the database, it revealed that DBY is an ARE-containing mRNA with class I ARE in the 3’UTR. We tried to realize whether ARE in the 3’UTR regulates DBY translation. By RNA-EMSA, UV-crosslink, and biotin pull-down experiments, we found Lethal Embryonic Abnormal Vision (ELAV)-like protein HuR, an ARE-binding protein, bind the 3’UTR of DBY in the region of 406-799th nucleotide. We analyzed the effect of HuR on posttranscriptional control of DBY using luciferase reporter assay system, and found that binding of HuR to DBY 3’UTR inhibits DBY protein expression without altering mRNA level.
In conclusion, our results revealed that HuR binds to the long form 3’UTR, mediating translational suppression but not affect mRNA degradation of DBY.
ACKNOWLEDGEMENT……………… ……………………… v

1.1 Definition and prevalence of male infertility 1
1.2 Causes of male infertility 2
1.3 Y chromosome and male infertility 4
1.4 The AZFa candidate gene, DEAD box on Y (DBY) 7
1.5 Regulation of gene expression in Eukaryotes 8
1.6 The role of 5’UTR of in translational regulation. 8
1.7 The role of 3’UTR in post-transcriptional regulation 10
1.8 HuR plays an role in mRNA stability and translational inhibition 11
1.9 Objective of this study 13

2.1 Total RNA isolation 14
2.2 Reverse transcriptional polymerase chain reaction (RT-PCR) 15
2.3 Rapid amplification of cDNA 5’ends (5’RACE) 16
2.4 Polymerase Chain Reaction (PCR) 18
2.5 Plasmid DNA preparation and DNA purification 19
2.5.1 Minipreparation of plasmid DNA 19
2.5.2 Minipreparation of plasmid DNA 19
2.5.3 Midipreparation of plasmid DNA 20
2.5.4 Gel extraction 21
2.5.5 PCR clean-up 22
2.6 Cell culture 23
2.6.1 Routine maintain and subculture 23
2.6.2 Cell stock 24
2.7 Computational analysis 24
2.8 In vitro synthesize radiolabled RNA probe 25
2.9 RNA electrophoretic mobility shift assay (RNA-EMSA) 26
2.10 UV cross-linking 27
2.11 Competition assay and Supershift 28
2.12 Biotin pull down assay 29
2.12.1 Synthesis of biotin labeled RNA probes 29
2.12.2 Biotin pull down 30
2.12.3 Western blot analysis 31
2.13 Construction of reporter plasmid with L1 sequence of human DBY 3’UTR 32
2.13.1 Vector preparation 32
2.13.2 Insert preparation and construction 33
2.14 Transient transfection and luciferase reporter assay 34
2.14.1 Transient transfection 34
2.14.2 Luciferase reporter assay 35
2.15 Real-time PCR 36

3.1 Identification of alternative DBY 5’UTRs in human testis 38
3.2 Expression pattern of untranslated regions of DBY gene in various human tissues 38
3.3 Identification of interacting protein of DBY 3’UTR 39
3.4 HuR binds the L1 region of DBY 3’UTR 40
3.5 HuR suppresses DBY translation in vivo 41

HuR and AU-rich element in post-transcriptional regulation 42
HuR cooperates with other proteins to regulate gene expression 44
References 46
Table 54
Figure 57
CURRICULUM VITAE………………………………………... 67
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