近日，Nucleic Acids Research雜志在線發(fā)表了中科院上海生科院植物生理生態(tài)所徐永鎮(zhèn)研究組關(guān)于ATP水解酶Prp28調(diào)控RNA剪接保真性機制的研究成果,。
內(nèi)含子的去除由RNA剪接催化完成,，是基因表達過程中一個重要的環(huán)節(jié)。內(nèi)含子序列包含三個保守的RNA區(qū)域：5’剪接位點（splicesite,，SS）,、剪接支點和3’剪接位點。這些序列有一定的保守性（GU—A—AG）,，但同時又有很大的靈活性,。剪接體是一個動態(tài)的大分子RNA-蛋白復合物，它的組裝和催化涉及到復雜的RNA-RNA和RNA-蛋白相互作用,，這些過程中發(fā)生的微小錯誤都可能使內(nèi)含子不被正確識別,，從而導致剪接紊亂，發(fā)生堿基插入,、刪除或移碼突變等錯誤,。已知15%以上的人類疾病是由于RNA剪接紊亂導致的，因此RNA剪接保真性（splicingfidelity）機制研究具有重要的意義,。屬于ATPase/RNAhelicase的一類剪接因子利用水解ATP的能量,，解開RNA-RNA雙螺旋結(jié)構(gòu)或移除與RNA相互作用的蛋白，促進剪接體組裝過程中的構(gòu)象重排,，研究人員這些ATP水解酶都參與了RNA剪接保真性的調(diào)控,。
盡管已有的遺傳學證據(jù)表明ATP水解酶Prp28在剪接體形成過程中促進了5'SS–U1snRNA雙螺旋結(jié)構(gòu)向5'SS–U6snRNA雙螺旋結(jié)構(gòu)的轉(zhuǎn)換過程，但是Prp28是否參與調(diào)控了5'剪接位點區(qū)域的保真性尚不清楚,。徐永鎮(zhèn)課題組通過一系列的遺傳學篩選,，分離得到了能夠提高次優(yōu)5'剪接位點底物（suboptimal5’SSsubstrate）剪接效率的prp28突變體，證明Prp28能夠調(diào)控內(nèi)含子5'SS的剪接保真性,。更為重要的是發(fā)現(xiàn)Prp28的調(diào)控功能只與5'SS:U6雙螺旋結(jié)構(gòu)穩(wěn)定性有關(guān)，而與5'SS:U1雙螺旋結(jié)構(gòu)穩(wěn)定性無關(guān)。進一步的研究表明Prp28通過ATP水解促進的構(gòu)象變化與5'SS–U6snRNA的配對速率相互競爭,，與U6snRNA配對速率相對較慢的次優(yōu)5'SS底物的剪接過程可以通過降低Prp28的水解酶活力得到恢復,。此外，通過在酵母和果蠅S2細胞中的報告基因檢測證實Prp28的E404殘基對剪接效率的維持也至關(guān)重要,，首次揭示了ATP水解酶的核心結(jié)構(gòu)域linker序列具有調(diào)節(jié)剪接保真性和剪接效率的雙重功能,。
該研究主要由博士生楊斐在徐永鎮(zhèn)研究員的指導下完成，受到中科院,、國家自然基金委和科技部的資助,。（生物谷Bioon.com）
Prp28調(diào)控5'SS剪接保真性的動力學模型
doi: 10.1093/nar/gkt149
Splicing proofreading at 5′ splice sites by ATPase Prp28p
Fei Yang,Xiu-Ye Wang,Zhi-Min Zhang,Jia Pu,Yu-Jie Fan,Jiahai Zhou,Charles C. Query and Yong-Zhen Xu
Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5′ splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5′SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5′SS. Prp28p is thought to facilitate the disruption of 5′SS–U1 snRNA pairing to allow for 5′SS–U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5′SS proofreading by Prp28p is dependent on competition with the stability of the 5′SS:U6 duplex, but not the 5′SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5′SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5′SS.
