RNA干擾現象對含Alu序列基因表現之影響

Abstract

人類基因體中有42%以上是由反轉錄跳躍子組成，其中數量最豐富的是屬於short interspersed element (SINE)家族的Alu序列。Alu序列長度約250-300bp，是由兩個被A-rich序列分隔的同源單體串聯排列組成，常出現在基因體中的intron及基因間區域，有時也會插入3’ untranslated region (UTR)，而目前Alu序列的研究主要集中在它的分類以及與遺傳疾病的關係。 RNA 干擾現象（RNAi）能引發基因轉錄後的表現下降，在自然界中主要和胚胎發育、宿主細胞防禦還有異染色質的形成有關。RNAi是由可和細胞內源的mRNA互補之雙股RNA所引發，它們在細胞內被切成小片段RNA後，進而造成mRNA的降解或轉譯的抑制。RNAi已經成為研究基因功能廣泛使用的工具。 我的論文主要是研究Alu序列是否也有扮演基因調控的角色。我們建構了pDPAlu95，它可以利用H1與U6兩個啓動子表達雙股Alu RNA，而綠螢光蛋白則當作報導基因，我們以螢光顯微鏡、西方點墨法、反轉錄聚合酶鏈鎖反應（RT-PCR）偵測轉染載體後細胞內GFP表現的程度。當pDPAlu95和基因下游接上完整Alu序列或含有完整Alu序列的3’UTR（來自六個不同的人類基因）之GFP載體共轉染至HEK293細胞，GFP的表現有顯著的下降，這暗示Alu序列可作為RNAi的標的序列而使基因轉錄後表現受調控。在另一個實驗，則顯示單一Alu單體也能夠成為標的序列而引發RNAi。 為了進一步分析Alu引發的RNAi抑制，我們將接上含有Alu序列的3’UTR（來自OACT1基因）之GFP載體中的Alu序列全部或部分剔除。pDPAlu95對3’UTR含有5’或3’ Alu單體的GFP載體表現仍有抑制的能力。不過，當GFP載體的3’UTR之Alu序列完全被剔除後，Alu序列仍能引發明顯地GFP表現抑制，這暗示OACT1的3’UTR中有可以作為pDPAlu95引發off-target抑制之潛在標的序列。 為了檢測5個內源性於3’UTR含有Alu序列的基因表現是否會被Alu引發的RNAi抑制所影響，我們也將pDPAlu95直接轉染至HEK293細胞株，並以RT-PCR偵測基因表現的情形，結果pDPAlu95卻無法抑制偵測的基因之表現，這和之前我們在pH1-siAlu95的實驗中所觀察的結果不大相符。所以，研究在細胞內Alu序列是否能引發RNAi的抑制時，選擇適當的表達載體可能是重要的

Retrotransposable elements, or retrotransposons, constitute about 42% of the sequence content of the human genome. The most abundant retrotransposons belong to the short interspersed element (SINE) family represented predominantly by the Alu sequences of 250-300bp long, which are composed of two homologous units in tandem separated by a stretch of AT-rich sequence. The Alu sequences are found commonly in introns and intergenic regions. In some occasions, they are present also in the 3’ untranslated region (UTR) as a consequence of retrotransposition. The current research of the Alu element is focused mainly on its taxonomical classification and relationship to genetics diseases. It has been known that down-regulation of expressed genes can be mediated by RNA interference (RNAi). This post-transcriptional event has been associated with embryonic development, host defense and chromatin remodeling. The mode of RNAi action is initiated by double-stranded (ds) RNA complementary to endogenous mRNA and the intracellularly processed small interfering RNA then triggers mRNA degradation or translational arrest. RNAi has become an important tool widely used for gene function analysis. The principal aim of my research is to examine whether the Alu sequence may play a biological role in gene regulation. We have constructed pDPAlu95 expressing ds-Alu RNA under the H1 and U6 double promoters. The green fluorescence protein (GFP) gene was used as a reporter to determine its expression level in transfected cells by the fluorescence microscopy, Western blotting, and the reverse-transcription polymerase chain reaction (RT-PCR). The expression of GFP were significantly reduced in cells co-transfected with pDPAlu95 and the GFP reporters fused downstream with a full-length Alu sequence only or with Alu-containing 3’UTRs derived individually from six different human genes. These results suggest that the Alu sequence may serve as a target for RNAi in post-transcriptional gene regulation. Additional studies showed that a single unit of the Alu sequence was sufficient for the RNAi targeting. To further analyze the Alu-mediated RNAi effect on the expression of GFP fused with Alu-containing 3’UTRs from the OACT1 gene, we generated a series of constructs with the Alu sequence completely or partially deleted. The suppressive activity on GFP expression conferred by pDPAlu95 was unaltered in constructs containing either the 5’- or 3’- unit of the Alu sequence in the 3’UTR. However, Alu-mediated reduction of GFP expression remained significant in the fusion gene completely devoid of the Alu sequence; suggesting the presence of potential off-targeting site(s) in the 3’UTR. We also examined the Alu-mediated RNAi effect on the expression of five endogenous genes that contain Alu sequences in the 3’UTR by transfecting pDPAlu95 directly into the HEK293 cell line. The expression level was assayed by the RT-PCR. Contrary to our early observations with pH1-siAlu95, pDPAlu95 failed to repress the expression of the five genes examined in this study. It is possible that selection of appropriate expression vectors may be critical to the in vivo study of Alu-mediated gene silencing by RNAi.