2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656923摘要：微小核醣核酸 (mircoRNAs) 是微小單股具調節性的核醣核酸，通常在後轉錄階段(post-transcriptional level) 控制基因的表現。微小核醣核酸影響非常廣泛層面的生物作用機制，他們的功能缺失可導致許多人類疾病與癌症。因此，了解微小核醣核酸調控基因的功能機制是分子生物學上相當重要的課題。而在微小核醣核酸生合成及作用時，許多步驟中發生了核醣核酸分子間或本身分子內交互作用的變化，以及核醣核酸蛋白複合體的構型重組，可以預期相當可能有核醣核酸解旋酶 (RNA helicase) 活性的參與，但是目前對此所知甚少。在真核生物中，核醣核酸解旋酶通常屬於 Superfamily 2 (SF2) 分類，尤其多屬於 DExD/H-box 核糖核酸解旋酶家庭。研究顯示 DExD/H-box 核糖核酸解旋酶可能跟細胞中幾乎所有牽涉到核醣核酸的生物作用機制皆有相關，在本計劃中我們想要更加了解 DExD/H-box 核糖核酸解旋酶在微小核醣核酸生合成及作用時所可能扮演的角色。微小核醣核酸 let-7 控制線蟲 C. elegans 的細胞週期與細胞分化，它的溫度敏感突變基因let-7(n2853) 可導致在進入成蟲階段時表皮接縫細胞 (seam cells) 無法脫離細胞週期而重複其在幼蟲階段的細胞分裂，以及生殖孔發育不良導致蟲體由生殖孔爆裂的性狀。對於let-7(n2853) 突變性狀的抑制子 (suppressor) 的遺傳分析可以用來尋找其他在let-7 作用機制中可能的參與因子。而另一個性狀輕微的突變基因 let-7(mg279) 則可以被用來進行合成致死篩選 (synthetic lethal screen) 而尋找 let-7 作用機制中的參與因子。在本計劃中，我們將利用對於let-7(n2853) 及 let-7(mg279) 的遺傳分析來篩選出可能與let-7 作用相關的 DExD/H-box 核糖核酸解旋酶，並進一步探討它們在微小核醣核酸生合成及作用階段中所可能扮演的角色。這項計畫的主要目標如下：目標一：鑑定與微小核醣核酸功能相關的 DExD/H-box 核糖核酸解旋酶目標一之一：利用小核醣核酸干擾 (RNAi) 篩選出可能與let-7 作用相關的 DExD/H-box 核糖核酸解旋酶目標一之二：確認候選 DExD/H-box 核糖核酸解旋酶與let-7 作用確有相關目標二：探討 DExD/H-box 核糖核酸解旋酶在微小核醣核酸功能中扮演的角色目標二之一：判斷候選 DExD/H-box 核糖核酸解旋酶是否與微小核醣核酸的生合成有關目標二之二：判斷候選 DExD/H-box 核糖核酸解旋酶是否調節微小核醣核酸的作用功能目標三：鑑定並分析與微小核醣核酸功能相關之 DExD/H-box 核糖核酸解旋酶的輔助因子完成此計畫能讓我們進一步了解微小核醣核酸的作用機制。由於微小核醣核酸調節幾乎所有生物作用機制中的基因表現，其缺陷可導致人類癌症，我們的研究結果將有貢獻於了解微小核醣核酸在癌症發生時所扮演的角色，亦有助於未來的癌症研究。<br> Abstract: MicroRNAs (miRNAs) are small non-coding regulatory RNAs that usually regulate gene expression at the post-transcriptional level. MiRNAs are involved in a broad spectrum of biological processes and dysfunction of miRNAs is associated with a variety of human diseases and cancers. Therefore, understanding the functional mechanisms of the miRNA pathway has become of great interest for investigation. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the expectation of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA, from transcription to RNA decay. Here we wish to better understand the roles of DExD/H-box RNA helicases in miRNA biogenesis and function.In C. elegans, the let-7 miRNA controls the timing of cell cycle exit and terminal differentiation. Its temperature-sensitive mutant allele let-7(n2853) causes heterochronic mutant phenotypes, such as reiterated lateral seam cell division and lethal vulval bursting at larva-to-adult switch. Genetic analysis of suppressors of let-7(n2853) could reveal functioning factors in the let-7 miRNA pathway. A weak allele let-7(mg279), without severe vulval bursting phenotype, could be used as a sensitized background in which to detect other defects in the miRNA pathway. Thus a synthetic lethal screen of let-7(mg279) could be used to identify functional factors in the miRNA pathway. In this project, we plan to use a reverse genetic screen based on RNA interference (RNAi), in the background of the alleles let-7(2853) or let-7(mg279), to identify novel DExD/H-box RNA helicases that are involved in the miRNA pathway. We will also further determine their possible roles in miRNA biogenesis or function. The specific aims for this proposal are:Aim 1. To identify DExD/H-box RNA helicases involved in miRNA functionAim 1.1. To screen for C. elegans DExD/H-box RNA helicases involved in the let-7 miRNA function by RNAiAim 1.2. To verify the candidate DExD/H-box RNA helicases involved in the let-7 miRNA functionAim 2. To determine the roles of candidate DExD/H-box helicases in miRNA functionAim2.1. To determine whether the candidate DExD/H-box RNA helicases are involved in miRNA biogenesisAim2.2. To determine whether the candidate DExD/H-box RNA helicases modulate miRISC activityAim 3. To identify and characterize protein cofactors of DExD/H-box RNA helicases functioning with miRNAsAccomplishment of this project will advance our knowledge on the mechanisms of miRNA action. Since miRNAs regulate gene expression in almost all cellular processes and their defects are usually linked to cancers, our results will also shed light on the roles of miRNAs in tumorigenesis and impact on future cancer research.The DExD/H-box 核醣核酸解旋酶微小核醣核酸let-7線蟲The DExD/H-box RNA helicasesmicroRNAlet-7C. elegans