MAC3A 與 MAC3B 在不同溫度下調控開花時間的研究

Abstract

選擇性剪接在植物感應溫度的調控中扮演非常重要的角色，而MOS4相關複合物或稱作MAC可作為剪接因子，去調節選擇性剪接同功型的產生。MAC的功能中心是由MAC3A與MAC3B這一組序列相近的E3 泛素連接酶所組成，它們具有調控剪接體和選擇性剪接的功能，所以MAC3A與MAC3B的突變會導致植物體內有大量的內含子保留事件的發生，而造成許多性狀。其中，mac3a mac3b突變體會對生物時鐘以及開花時間造成影響，然而，MAC3A與MAC3B所參與的詳細途徑以及調控機制仍未明瞭。在本論文中，我研究了 MAC3A/MAC3B 是否透過調節選擇性剪接影響溫度調控的開花途徑。我的實驗結果顯示，當MAC3A與MAC3B產生缺陷時，植物會降低感應溫度變化而調控開花時間的能力，其中可能造成的原因是因為在mac3a mac3b中，開花調控因子FLM的同功型無法像在Col-0隨著溫度而產生改變。此外，溫度也會影響植物生物時鐘的功能，而進一步影響下游的光週期所調控的開花途徑，先前研究證實在mac3a mac3b中，有兩個生物時鐘相關的基因，PRR7與PRR9會額外產生大量的內含子保留事件。在我的實驗結果顯示，溫度的升高下，PRR7的同功型原本會隨之上升，然而此一現象並無發生在mac3a mac3b中。我進一步探討溫度如何調控MAC3A和MAC3B，我調查了在MAC3A與MAC3B 5’端非轉譯區域(5’ untranslated region)中的上游開放讀序 (upstream open reading frame, uORF)，此段調控序列被認為可以抑制下游的編碼區，而且與環境的刺激息息相關。藉由冷光素酶的報導試驗，我確認了MAC3A與MAC3B的uORF確實能夠抑制下游編碼區的轉譯，而且溫度的變化會藉由uORF影響其下游的轉譯。總結所有結果，MAC3A與MAC3B可能會藉由影響FLM以及PRR7的選擇性剪接參與開花時間的調控，而且MAC3A與MAC3B的uORF會受到溫度的調節，影響下游基因的轉譯活性。這可能是導致FLM的選擇性剪接為何會因應溫度變化改變的因素之一。

Alternative splicing (AS) plays a major role in regulating plant thermal adaptation pathways. The MOS4-associated complex (MAC) acts as a splicing factor to interact and regulate spliceosome, which then mediates the production of alternative splicing isoforms. E3 ubiquitin ligases MAC3A and MAC3B are the functional components of the MAC to modulate splicing. Mutation of them causes an increase in intron retention events, which affect pleiotropic biological processes, including circadian rhythm and flowering time. However, the mechanisms that MAC3A/MAC3B regulate these pathways remain to be elucidated. In this study, I examined the roles of MAC3A/MAC3B in mediating AS to regulate temperature-dependent flowering. The defects in both MAC3A and MAC3B reduced the sensitivity in the temperature-responsive flowering. This phenomenon may be partially explained by alterations in the ratios of flowering regulator FLM splicing isoforms in the mac3a mac3b at ambient temperatures. Additionally, temperatures also affect the circadian clock to further mediate downstream flowering pathways. Here, I found that elevated temperatures induced intron retention of PRR7, and these phenomena did not occur in mac3a mac3b. This alteration could affect the photoperiodic flowering pathways and lead to less sensitivity in the temperature-responsive flowering in mac3a mac3b. To further investigate the potential translational mechanism that MAC3A/ MAC3B responded to temperatures, I examined the upstream open reading frames (uORFs) in their 5’ UTR. uORFs may repress the translation of downstream coding regions and are reported to respond to environmental stimuli. The results from luciferase reporter assays suggested that the uORFs in MAC3A/3B serve as temperature-responsive translational suppressors. To sum up, my results indicated that MAC3A/MAC3B may regulate the AS of FLM and PRR7 to engage in flowering time control, and the uORFs of MAC3A/MAC3B are temperature-responsive translational repressors. These findings may explain the temperature-responsive AS of FLM.