探討酵母菌環形核糖核酸與其在壓力環境下的表現

Abstract

隨著對轉錄體的了解與研究，許多不會轉譯出蛋白質的非編碼核糖核酸逐漸被發現，其中包含了環形核糖核酸(circular RNAs, circRNAs)。基因從DNA經轉錄(transcription)形成pre-RNA後，會被剪接(splicing)成線型的RNA。然而circRNAs 的生成是透過反式剪接(back-splicing)形成環狀構造。因為其封閉環狀的構造，RNA外切酶無法有效地作用，因此這類的RNA在細胞內相對穩定且不易被降解。circRNAs最早在1976年於植物的類病毒中發現，隨著定序技術與生物資訊分析的方法的進步，在許多物種都有發現circRNAs，包括了人類、植物以及真菌。circRNAs已知的功能如下：可作為miRNA或RNA-binding protein sponge、調控基因轉錄、參與細胞應對壓力反應的調控。然而因為circRNAs的構型特殊且表現量較少，在高等真核生物中研究circRNAs 的功能仍然相當困難。因此我的目標是在透過研究酵母菌(Saccharomyces cerevisiae)中的circRNAs，進一步了解他們的功能。 酵母菌中有10個基因擁有兩個Intron，先前的研究針對這十個基因進行驗證，並報導了其中六個基因會生成circRNAs。為了更全面的探討酵母菌的circRNAs，我利用核糖核酸測序(RNA-seq)研究酵母菌circRNAs，使用偵測程式進行circRNAs的搜尋，包括了CIRI、find_circ以及NCLscan，並輔以針對性的back-splicing 序列模板進行比對，用於驗證酵母菌的circRNAs。分析指出circEFM5是酵母菌中表現最多的circRNAs，為了進一步了解circRNAs的在壓力調控中的功能，我將酵母菌培養在一系列的壓力環境中，包含了生長至靜止期(Stationary phase)、溫度變化、高濃度的鹽類、金屬離子、氧化逆境，以及缺乏氮的環境，並觀察circEFM5與其直線型RNA在細胞內的比例變化。透過RT-qPCR的結果可以觀察到EFM5環形線型的比例在不同的逆境下有所改變，暗示著circEFM5 可能會參與酵母菌的壓力反應調控。在未來希望能透過全面性分析酵母菌的circRNAs、探討其在壓力環境下的轉錄體，進而了解circRNAs的分子功能與在調控壓力反應中所扮演的角色。

CircRNAs is a unique class of non-coding RNA revealed from explosion of transcriptomic analyses. Viroid was the first circRNA discovered in 1976. With the advance of sequencing and bioinformatic analysis, more circRNAs were identified across species, from fungi, plant to human. Different from canonical splicing, CircRNA is generated through back-splicing, in which the downstream splice site of an exon is joined to its upstream splice site. This ultimately forms a closed loop structure with 3' to 5' at the junction. CircRNAs can function as miRNA sponge, regulation of transcription and involving in stress responses. However, the functional study of circRNA remains highly challenging in higher eukaryotes because of its unique structure, low abundancy, I aimed to address this issue by studying circRNAs in the budding yeast (Saccharomyces cerevisiae), which is a powerful and well-established model system. Previously, ten genes in yeast with two introns were surveyed and six of them were found to produce circRNAs. To investigate yeast circRNAs in a more comprehensive way, I have established a platform to study yeast circRNAs by RNA-seq. CircRNAs detection algorithms such as CIRI, find_circ, and NCLscan combined with customized templates were used to identify circRNAs in yeast. CircEFM5 was reported to be the most abundant circRNAs and was selected for further investigation under a variety of stress conditions such as stationary phase, temperature shift, high concentration of salt or metal, nitrogen starvation, and oxidative stress. By using RT-qPCR, I observed that the circular/linear ratio of EFM5 changed upon certain stresses, implicating that circEFM5 might be regulated upon stresses. My long-term goal is to comprehensively document yeast circRNAs and take the advantage of yeast genetics to investigate their functions in response to stresses in details.