前剪接體形成之分子機制

Abstract

前驅訊息核糖核酸 (pre-mRNA) 剪接反應是由U1結合在5端剪接位，以及Msl5-Mud2異二聚體與分歧點 (branchpoint) 的結合而開始的，從而形成委任複合體 (Commitment complex)。接著由DEAD-box蛋白Prp5協助U2 小核醣核酸複合體與前驅訊息核糖核酸的結合，形成前剪接體。在形成剪接體的過程中，U2 如何被引導至前驅訊息核糖核酸，如何替換Msl5-Mud2，以及Prp5如何脫離的詳細機制尚不清楚。在先前的研究中，我們透過使用分歧點突變的前驅訊息核糖核酸，發現一個含有含有Prp5新的中間複合體，FIC (Prp five-associated intermediate complex )。在此篇研究中，藉由免疫沉澱與化學交叉鏈結分析，我發現Msl5仍然與FIC結合，並且主要的結合位置為分歧點上游的隱蔽結合位 (CBS, Cryptic binding site)。在FIC上，U2 並未與突變的分歧點形成鹼基配對，反而是與分歧點下游區域形成配對。CBS的存在與其相對於分歧點的位置，對於委任複合體的形成非常重要，CBS的缺失也會影響U2與前驅訊息核糖核酸的結合。在剪接反應中使用能夠與突變分歧點完整互補的突變U2小核醣核酸，我發現恢復U2與突變分歧點之間的鹼基配對可促進Prp5從FIC分離，並且挽救剪接缺陷。當我將分歧點下游的序列進行點突變，並增強此區域與U2的配對強度，會降低剪接反應的速率。此結果暗示著在前剪接體形成的過程中，U2可能被加載到分歧點的下游，並沿著3端至5端的方向掃描前驅訊息核糖核酸來搜索分歧點。本研究的結果顯示，Msl5與前驅訊息核糖核酸的正確相互作用對於U2的結合很重要，我也發現Prp5可能是由於U2與分歧點進行鹼基配對後，U2的結構變化而分離，進而推動後續剪接反應的進行。

Splicing of pre-mRNA is initiated by binding of U1 snRNP to the 5’ splice site and of Msl5-Mud2 heterodimer to the branch site (BS), together forming the commitment complex (CC). Subsequent binding of U2 snRNP to the pre-mRNA, facilitated by DEAD-box protein Prp5, forms the prespliceosome. How U2 is recruited to the pre-mRNA to replace Msl5-Mud2 and what triggers dissociation of Prp5 during prespliceosome formation is unknown. The research group of Dr. Soo-Chen Cheng previously identified a prespliceosome precursor complex using BS-mutated ACT1 pre-mRNA that causes arrest of Prp5 on the spliceosome, forming a complex called FIC (for Prp five-associated intermediate complex). By immunoprecipitation and cross-linking analyses, I show that Msl5 stalls on the FIC and binds mainly at the upstream cryptic branch site sequence (CBS). In contrast, instead of interacting with the BS, U2 interacts with sequences downstream of it. Deletion of the CBS weakens binding of Msl5 and U2 to BS-mutated pre-mRNA, and the relative position of the CBS to the mutated BS is important for recruitment of U2 snRNP. Restoring base-pairing between U2 and the mutated BS promotes dissociation of Prp5 from the FIC and rescues the splicing defect in a CBS-dependent manner. Mutating sequences downstream of the mutated BS to enhance the pairing strength between U2 and the pre-mRNA reduced the rate of the splicing reaction, suggesting that U2 may be loaded downstream of the BS and then seeks the BS along the pre-mRNA. My results demonstrate that proper interaction of Msl5 with the pre-mRNA is important for U2 recruitment, and that Prp5 dissociates possibly due to a conformational change of U2 snRNA after base-pairing with the BS to drive the reaction forward.