| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 94 === 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.
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