線蟲體內微小核醣核酸let-7之專一性結合蛋白鑑定

Abstract

microRNAs在10幾年前被發現以來已經有相當多的研究在探討其調控細胞基因表現的機制，但是關於microRNAs本身是如何作用、有甚麼共同因子一起促進其作用或抑制其作用，至今還是有許多地方尚未釐清。在我的論文中主要是想要在C. elegans體內探討let-7上除了目前已知的一些RISC組成蛋白與其結合之外，是否還有其他蛋白質會與let-7專一性地結合，再進一步探討它們是如何參與在let-7調控基因表現的過程。我的論文策略主要是想利用2’-O-methyl oligo- nucleotides pull-down assay來探討let-7上是否有專一性的結合蛋白。原理是利用人外加標定有biotin的let-7 antisense序列到線蟲細胞萃取液中去辨認內生性的let-7，再用帶有Streptavidin的基質將內生性的let-7及其結合蛋白分離出來並利用一維以及二維蛋白質電泳分析其蛋白質成分。 針對所有被antisense let-7 oligonucleotide純化出來的候選蛋白質，我們利用RNAi降低其基因表現並觀察其產生的性狀，rpa-1以及mtr-4在RNAi處理後會出現col-19表現量降低、vulva 發育缺失、seam cell分化異常、alae斷裂等典型的let-7功能不全時所會看到的型態學變化。進一步的研究發現抑制mtr-4的表現會造成mature let-7量下降、pri-let-7以及pre-let-7量增加的情形，代表著mtr-4可能參與在let-7生合成過程中。然而抑制rpa-1的表現對於let-7的生合成則沒有影響，是否rpa-1在let-7執行功能時一起參與其中則是還要再進一步去證實。

MicroRNAs are a class of small non-coding RNAs which regulate more than half of human genes by targeting to their complementary sites on mRNA 3’UTRs. MicroRNAs assemble with additional proteins into ribonucleopreotein complexes called miRNA-induced silencing complexes (miRISCs) and the composition of miRISCs is still not clear. Interestingly, in our previous study, we found that the let-7 miRNA and their family members, such as mir-84, can assemble into specific miRNPs in vitro. Here we aim to identify specific factors that associate with let-7. We use anti-sense oligonucleotides pull-down to isolate endogenous let-7 along with its associating proteins and then analyze the pull-down products by 1-D or 2-D protein electrophoresis. Further we use RNAi to reduce the gene expression of the candidate let-7-binding proteins and investigate their genetic interaction with let-7. Our results indicate that knock-down of rpa-1 or mtr-4, which encode candidate let-7-binding proteins found in our pull-down assay, resulted in let-7 defective phenotypes, like col-19 down- regulation, seam cell differentiation defects, alae formation defects and vulva protruding. We further found that knocking down mtr-4 caused accumulation of pri-let-7 and pre-let-7 but decrease of mature let-7 level. On the other hand, when we knocked down rpa-1, the level of let-7 was not altered. We hypothesize that MTR-4 may be involved in let-7 primary transcript processing or pre-let-7 exporting from the nucleus to the cytoplasm. The role of rpa-1 in the let-7 regulation pathway is still unknown and awaited to be further investigated.