線蟲DEAD-box核醣核酸解旋酶DDX-47及異質核醣核酸蛋白HRP-2/hnRNP Q在let-7 miRNA功能之角色

Abstract

微小核醣核酸(MicroRNAs or miRNAs)是一段約22個核苷酸長的non-coding RNA，主要是透過結合在目標基因的3端不轉譯區，抑制其轉譯或是造成mRNA降解來調控基因的表現。 在線蟲中，let-7 微小核醣核酸透過降低lin-41的表現調控線蟲從第四幼蟲期到成蟲的轉換。低量的 let-7 表現無法有效抑制lin-41，會造成線蟲上皮接縫細胞 (hypodermal seam cell) 終端分化遲緩及外陰發育不正常。表現let-7亞效等位基因的線蟲 (n2853) 通常會在幼蟲轉換至成蟲時，產生接縫細胞最終分化延遲以及外陰爆裂死亡的性狀。let-7 miRNA調控lin-41表現的現象在物種間具有保守性。在人類中，let-7低量表現或其調控的目標基因如LIN41/TRIM71及LIN28等致癌基因，則與許多癌症的發生有高度相關性。為了進一步釐清miRNA調控的機制，尤其是let-7對於lin-41的調控，我們試圖去找出可能參與let-7生合成或功能的DEAD-box 核醣核酸解旋酶蛋白，及異質核醣核酸蛋白Heterogeneous ribonucleoproteins (hnRNPs)。透過在線蟲內進行RNA干擾篩選實驗，我們發現降低核醣核酸解旋酶DDX-47以及異質核醣核酸蛋白HRP-2的表現，可以抑制let-7亞效等位基因線蟲 let-7(n2853) 異時基因異常的性狀，顯示此兩種蛋白可能與let-7的功能有關。在此，我們證實 DDX-47 影響核醣體核醣核酸的生合成，降低DDX-47表現導致核醣體壓力，可能引發其他非 let-7 調控路徑表現去抑制 let-7(n2853)突變株的性狀。另一方面，我們證實HRP-2可以與lin-41 之3端不轉譯區上一段腺嘌呤-尿嘧啶豐富序列結合。有趣的是，人類中HRP-2的同源蛋-hnRNP Q也會與TRIM71/LIN41的3端不轉譯區上腺嘌呤豐富序列有交互作用。去除掉hnRNP Q的結合位可以提高let-7抑制的現象，根據結果顯示出此具有物種間保守性的hnRNP可能透過介入miRNA和3’UTR間的交互作用來調控lin-41表現。

MicroRNAs (miRNAs) are ~22-nt-long small non-coding that usually regulate gene expression by binding to the 3’UTRs of target mRNAs and triggering translational repression and/or mRNA degradation. In C. elegans, the let-7 miRNA down regulates lin-41 that controls transition from the 4th larva stage to adults. Low levels of let-7 cause inefficient repression of lin-41, leading to attenuated terminal differentiation of hypodermal seam cells and abnormal vulval morphorgenesis. Animals carrying a hypomorphic let-7(n2853) allele exhibit reiterated division of seam cells and die by bursting through the vulva. The regulation of lin-41 by let-7 is conserved across species. In humans, serveral let-7 targets, including LIN41/TRIM71 and LIN28, are oncogenic and low expression of let-7 has been shown highly correlated to a variety of cancers. To better understand miRNA-mediated regulation, the regulation of lin-41 by let-7 in particular, we sought to identify novel effecting factors from the DEAD-box RNA helicase family and heterogeneous nuclear ribonucleoproteins (hnRNPs) for let-7 biogenesis and/or function. By candidate-based RNAi screens in C. elegans, we have found that depletion of the RNA helicase DDX-47 or the hnRNP HRP-2 suppressed the heterochronic phenotypes caused by let-7(n2853), suggesting that these proteins may function with let-7. Here, we show that DDX-47 affects ribosomal RNA biogenesis and may suppress the let-7 heterochronic phenotypes through a parallel pathway, perhaps induced by nucleolar stress. HRP-2 binds to an AU-rich element of the lin-41 3’UTR. Interestingly, the human HRP-2 homolog, hnRNP Q, also interacts with the TRIM71/LIN41 3’ UTR through binding to an A-rich region. Deletion of this putative hnRNP Q binding site enhances repression by let-7, suggesting a role of this conserved hnRNP in the regulation of lin-41 through miRNA-3’UTR interaction.