- 學位論文
秀麗隱桿線蟲重要 Argonaute 蛋白 CSR-1 其蛋白質結構及結構域功能之探討
Protein structure and domain function of the essential C. elegans Argonaute protein CSR-1
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摘要
Argoanute蛋白是一個高度序列保守的蛋白質，它被廣泛地發現在不同生物體之間，包含了人類、果蠅、線蟲以及細菌。而做為一個在RNA干擾的過程裡，於RNA induced silencing complex (RISC)當中扮演重要的功能性角色，Argoanute在秀麗隱桿線蟲之中有超過20個蛋白質家族成員，它們分別都在小RNA pathway裡面扮演著不同功能的角色。而當中，CSR-1，一個隸屬於秀麗隱桿線蟲的次級Argoanute蛋白，它在近年來科學家們廣泛的研究中被認為是線蟲當中相當重要的蛋白，特別的是它可以進入細胞核中去調控其相關的基因的表現量，甚至會去影響到了染色體分離的過程，這些功能都說明了CSR-1對於秀麗隱桿線蟲的生殖能力的重要性，當CSR-1突變時，會導致線蟲有嚴重的不孕情形產生。然而對於CSR-1蛋白質本身的結構功能上的理解還並未透徹。 因此，在這篇研究裡，我們利用昆蟲細胞以及昆蟲桿狀病毒去製造CSR-1a的重組蛋白並加以純化，期望能夠利用冷凍電子顯微鏡的方式將此重組蛋白進行蛋白質結構的分析。 我們也進一步的製造出了蛋白質結構域置換的基因改造線蟲，利用製造額外一條CSR-1a的基因並加以改造成帶有WAGO-1的PAZ結構域的重組基因，WAGO-1是線蟲中具有抑制基因表現的次級Argonaute蛋白，這樣子我們就可以順利的深入研究CSR-1的蛋白結構域對其功能的影響而不會影響到線蟲本身的生殖能力。 除此之外，我們也利用了近年來蓬勃發展的蛋白質結構預測的電腦軟體，將秀麗隱桿線蟲的CSR-1、WAGO-1以及CSR-1 (WAGO-1 PAZ)的蛋白質進行立體結構模擬分析，並且也進一步的進行蛋白質結構上的差異比對分析，從我們比對分析的結果，可以得知CSR-1相對於WAGO-1的結構上存在差異性，即便兩者同是隸屬於線蟲當中的次級蛋白質，藉由這樣的結果或許可以進一步的知曉並分析CSR-1為何如此的重要以及其特別的功能性。
並列摘要
Argonaute proteins are highly conserved across different species and play a crucial role in targeted mRNA decay through their association with small interfering RNAs, a process known as RNA interference. The Argonaute protein family is characterized by the presence of two domains: the PAZ domain and the PIWI domain. In the nematode Caenorhabditis elegans, there are over 20 different Argonaute proteins, with CSR-1 being the sole protein required for fertility and embryo survival. Unlike its conventional function in targeted mRNA degradation, CSR-1 is also involved in promoting the expression of its target genes by recruiting RNA polymerase II in the nucleus. Interestingly, CSR-1 exhibits two isoforms: CSR-1a, a longer isoform that regulates spermatogenic genes, and CSR-1b, a shorter isoform that regulates oogenic genes. The unique differences in CSR-1 compared to other gene silencing Argonautes have yet to be fully understood. In this study, our primary focus is to determine the three-dimensional protein structure of CSR-1a using cryo-electron microscopy (cryo-EM). To achieve this, we expressed recombinant CSR-1a protein utilizing the insect cell baculovirus system, following a previously established protocol. We also modified the purification step in order to get pure soluble recombinant protein. Additionally, we conducted domain swapping experiments by exchanging the PAZ domain of CSR-1a with the PAZ domain of WAGO-1. WAGO-1 is a secondary Argonaute protein known for its role in gene silencing. We introduced this chimeric variant of CSR-1a, which contains the PAZ domain of WAGO-1, into worms that carried a single-copy of CSR-1a on chromosome II. Through this approach, we were able to bypass the infertility phenotype caused by endogenous csr-1 mutation and successfully examine the expression of both CSR-1a protein and its chimeric variant. Furthermore, we employed computational methods to predict the protein models of various Argonaute proteins, including CSR-1a, WAGO-1, and CSR-1a (WAGO-1 PAZ). By comparing these predicted models, we observed distinct structural differences between CSR-1a and WAGO-1, providing intriguing insights into the diverse functions of these two Argonaute proteins with opposing roles.
參考文獻
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