阿拉伯芥中可能形成R-loop基因之探討

Abstract

R-loop為一RNA-DNA雜合體加上一單股DNA所形成的三股並存形式，在R-loop中，包含由轉錄後的RNA與模板DNA (template strand DNA) 重新結合形成之雙股雜合體，以及維持單股DNA之非模板DNA (non-template strand DNA)。R-loop會參與大腸桿菌及哺乳類粒線體的DNA複製、免疫球蛋白的 “類型轉換” (class switching)、阻礙轉錄的進行、以及影響基因組的穩定性。在阿拉伯芥中已發現第一個會形成R-loop的基因─COOLAIR，此基因會藉由R-loops的穩定度來調控基因的轉錄反應進而影響基因表現。為更進一步的探討植物中R-loop的形成是否是普遍存在的基因調控機制，本研究參考前人的實驗結果及Roy和Lieber所提出的quantitative model of R-loop forming sequence，用程式語言「C++」設計出用以搜尋可能形成R-loop的基因的生物資訊程式，篩選出53個阿拉伯芥基因具有形成R-loop潛力之基因，以gel mobility shift assay發現At1g61600、At1g75550、At1g77030以及LOB domain-containing protein 18 (LBD18) 於胞外進行轉錄後有DNA-RNA雜合體的存在，最後利用native bisulfite sequencing assay觀察到在細胞內LBD18會形成長度約為50 bps的R-loop結構，而R-loop的生成位置與程式預測相符，且其長度在基因表現量上升時也會增長。雖然R-loop與轉錄反應之間的關係還需要更多的實驗去探討，但本研究初步建立了由序列特徵搜尋有潛力生成R-loop基因的方法，並證實了在阿拉伯芥中除了COOLAIR外還有其他會形成R-loop的基因。

R-loop is a three strand nucleic acid structure consisting of an RNA-DNA hybrid plus a single-stranded DNA. R-loops are formed when newly synthesized RNA threads back to anneal with the template strand DNA, leaving the non-template strand DNA remaining as a single strand. R-loops are known to participate in various processes, including the replication of ColE1-type plasmids in Escherichia coli and mammalian mitochondrial DNA; class-switch recombination involved in the production of mammalian immunoglobulin; impairment of transcription elongation, or genome instability as results from a double-strand break of DNA. In Arabidopsis, the expression level of COOLAIR, the first gene discovered that can form R-loops in plants, is regulated by the stability of R-loops. Hence, the goal of this study was to explore if R-loop formation is a general gene regulatory mechanism in plants. First, an R-loop searching program written using C++ (with help from Dr. Chien-Yu Chen) was established based on a quantitative model of R-loop forming sequence proposed by Roy and Lieber. 53 genes were predicted to be able to form R-loops by the program. At1g61600, At1g75550, At1g77030, and LOB domain-containing protein 18 (LBD18) were selected to estimate and confirm their ability to form RNA-DNA hybrids in vitro using the gel mobility shift assay. The possible formation of R-loop in LBD18 in vivo was further investigated using the native bisulfite sequencing assay. The R-loops was formed in an up to 50 bp region that agrees with the prediction by the program. Further, the length of the R-loops increased to 130 bp when its gene expression was up-regulated by indole-3-acetic acid (IAA). In this research, we established a system to search R-loop forming genes based on sequence features and showed that there were other genes besides COOLAIR that could form R-loops in Arabidopsis.