探討MAC3A相互作用的蛋白質網絡

Abstract

選擇性剪接是一種重要的轉錄後調控機制，以調節基因表現來因應外在環境變動。在阿拉伯芥中，MOS4相關複合體(MAC)作為一個保守的剪接因子，參與mRNA的調控中。MAC的核心成員包括兩個E3泛素連接酶: MAC3A與MAC3B。前人研究表明，這兩者的雙突變體會導致全基因組內含子保留事件明顯增加。然而，MAC3A和MAC3B在植物中調節mRNA剪接的具體機制尚未明瞭。我們實驗室先前的研究發現，與Col-0相比，mac3a mac3b突變體在短日照條件下表現出明顯的早花外表型。此外，RNA定序分析發現，在短日照下，與Col-0相比mac3a mac3b中超過98%的剪接事件發生變化，特別是內含子保留事件。因此，我們假設MAC3A和MAC3B能透過與其他蛋白的相互作用共同調控選擇性剪接，從而影響短日照下的開花外表型。為了揭示MAC3A的相互作用蛋白質網絡並確認潛在的相互作用者，進一步探討其分子機制。在本篇論文中，我使用免疫沉澱串聯質譜分析(IP-MS)來鑑定MAC3A的相互作用蛋白，並研究這些相互作用如何調控剪接。我在阿拉伯芥中表達全長(35s::FLAG-MAC3A-FL)或E3連接酶缺陷（35s::FLAG-MAC3A-H31A/D34A）的MAC3A，並以mac3a mac3b作為背景對照進行IP-MS，藉由數據分析，我鑑定出264個與全長MAC3A相互作用的蛋白，以及811個與E3連接酶缺陷的MAC3A相互作用的蛋白。基因本體論(GO)分析顯示，這些蛋白與mRNA加工過程相關。在相互作用候選蛋白中，MAC的核心成員以及剪接體組成成員是顯著富集，表明MAC3A確實參與了剪接調控。最後，我挑選了幾個參與剪接和開花調控的相互作用候選蛋白，進一步驗證它們與MAC3A之間的相互作用，並探討它們在RNA剪接調控中的分子機制。

Alternative splicing (AS) is a critical post-transcriptional mechanism that modulates gene expression, serving as an effective means to respond to environments. The conserved Arabidopsis MOS4-associated complex (MAC) functions as a splicing factor and participates in mRNA splicing. MAC3A and MAC3B, the two U-box E3 ubiquitin ligases are core components of MAC, and their mutants have been reported to cause genome-wide intron retention (IR) defects. However, their roles in mediating mRNA splicing in plants remain unclear. Previous studies in our lab observed that the mac3a mac3b showed an early-flowering phenotype compared to Col-0 in short-day conditions. Furthermore, RNA-sequencing (RNA-seq) analysis revealed that more than 98% of splicing events, particularly IR events, were altered in the mac3a mac3b. Therefore, we hypothesize that MAC3A and MAC3B, along with other interacting proteins, mediate AS, influencing the flowering phenotype under short-day conditions. To uncover the MAC3A interacting protein network, identify potential interactors, and further explore the molecular mechanism, I used Immunoprecipitation-Mass Spectrometry (IP-MS) to identify MAC3A's interacting proteins and investigate their potential roles in modulating splicing. I performed IP-MS with Arabidopsis expressing full-length (35s::FLAG-MAC3A-FL) or E3 ligase-defective (35s::FLAG-MAC3A-H31A/D34A) MAC3A together with mac3a mac3b background control. Through analyses, I identified 264 and 811 interacting candidates with full-length and E3 ligase-defective MAC3A, respectively. Gene Ontology (GO) analysis indicated that these proteins are associated with mRNA processing. Among the interacting candidates, the major MAC subunits and spliceosome components were enriched, indicating that MAC3A is indeed involved in splicing regulation. Additionally, the ubiquitin ligases-defective MAC3A-H31A-D34A may not alter the core components of MAC or spliceosome interactions. Finally, I have selected several interacting candidates involved in splicing and flowering regulation to further verify the interactions and investigate their molecular mechanisms in regulating RNA splicing.