- 學位論文
響應環境變化而發生的截然不同的剪接調控
Distinct splicing regulation in response to environmental changes
摘要
剪接內含子是普遍存在的非編碼核糖核酸序列，通常注定要從新合成的核糖核酸中去除並迅速降解。但是，最近的研究表明，當營養缺乏時，積累的內含子可以幫助酵母細胞存活。在這裡，我們描述了一個指數，簡稱為IA，可用以評估內含子積累水平和剪接效率。通過使用IA指數的篩選準則，可以在各種生長條件下鑑定出新的內含子和信使核糖核酸前驅物積累事件。令人驚訝的是，在所有檢查的生長條件下，其中包括在營養培養基中指數生長期間，都發現了許多信使核糖核酸前驅物積累事件。內含子水平的提高是由於這些轉錄物不能被有效剪接和/或這些內含子不能被降解導致的。此外，在不同生長條件下，轉錄物的剪接效率會隨之變動，這表示剪接是響應於環境而專門進行調節的。順式信息分析表明，剪接效率低下的轉錄本內含子短，啟動子弱。剪接效率會隨轉錄加強而增加。為了確定反式因子與剪接事件之間的相互作用，我們開發了細胞內調節子的高通量篩選技術（CHRES），並且套用於約4,300種不同單基因缺失的酵母菌珠中。有趣的是，剪接較差的轉錄本的調節子與標準剪接的轉錄本幾乎不同。我們發現Ras / PKA信號傳遞路徑參與了低效率剪接轉錄本（KIN28）的調控。當將飢餓的細胞轉移到含有葡萄糖的豐富培養基中時，它可使KIN28轉錄物迅速被剪接，而當葡萄糖受到限制時，KIN28轉錄物則不能被有效剪接，這有助於細胞在需要呼吸作用時生長。因此，本篇研究展示了我們鑑定新型內含子積累事件及其調控機制的有效方法。同時展示了具有轉錄物特定性的調控網絡，其中包括營養刺激物的感知和信號傳遞讓剪接開始進行，使細胞得以快速適應新環境。
並列摘要
Spliceosomal introns are ubiquitous non-coding RNA sequences that typically destined for removing from newly synthesized RNAs and rapidly degrading. However, recent studies suggest that accumulated introns help yeast cells survive when nutrients were scarc. Here, we describe an index, IA, to evaluate intron accumulation level and splicing efficiency. By using IA criteria, novel intron and pre-mRNA accumulation events were identified in various growth conditions. Surprisingly, lots of pre-mRNA accumulation events were found in all examined growth conditions including during exponential growth in rich medium. Increased levels of intron result from failures of these transcripts to be efficiently spliced and/or of these introns to be degraded. In addition, the splicing efficiency of transcripts varies with different growth conditions that suggest splicing is specifically regulated in response to environments. Cis-information analysis demonstrated the inefficiently spliced transcripts have short intron and weak promoters. Splicing efficiency increases with increased transcription. To identify interactions between trans-factors and splicing events, we developed a cell-based high-throughput regulator screening (CHRES) and implemented in the background of ~4,300 different gene deletions. Interestingly, regulators of the poorly spliced transcripts are distinct from the canonically spliced transcript. The Ras/PKA signaling transduction was found to involve in the regulation of inefficiently spliced transcript KIN28. It enables KIN28 transcript to be rapidly spliced when shifting starved cells to rich medium containing glucose but to be inefficiently spliced when glucose is limited, that help cell in respiratory growth. Thus, our work demonstrates the capacity of our approach to identifying novel intron accumulation events and their regulatory mechanisms. It exhibited transcript-specific regulatory networks that include sensing and signaling of the nutrient stimuli to activate splicing, allowing cells to quickly adapt to the new environments.