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PNAS:冯英等RNA剪接调控研究获进展

来源:上海营养所 2013-07-24 23:40

近日,PNAS在线发表了中科院上海生科院营养科学研究所冯英研究组的最新研究进展:“Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression”。该研究揭示了RNA二级结构在剪接调控中的新机制,并首次证明了MYC调控蛋白FUBP1同样具有剪接调控活性。

RNA剪接是连接转录与翻译的重要桥梁,也是生物体蛋白质多样性的重要保证。在真核生物中,mRNA前体被剪接成为成熟的mRNA一般需要在多种蛋白质及snRNA的帮助下,经历两步转酯剪接反应来完成。目前对于第二步剪接反应的调控知之甚少。

冯英研究组在前期工作中发现了一种特殊的mRNA前体,该前体在剪接反应已经开启的情况下,仅将转酯反应进行到第一步即停止,第二步反应则被完全抑制。冯英研究员指导的博士生李璜等研究人员对造成这一特殊现象的机制进行了深入研究。利用多种体外、体内的剪接模型以及定点突变,研究组发现剪接位点附近形成的RNA二级结构会显著抑制第二步剪接反应的发生;同时还发现单链DNA结合蛋白FUBP1通过与剪接沉默子(ESS)元件结合,能够有效抑制第二步剪接反应的发生。在体外剪接环境中降低FUBP1蛋白水平,可以有效促进第二步剪接反应的效率;而在细胞水平上改变FUBP1的含量不仅会影响该mRNA前体的剪接,还会改变一系列内源mRNA前体的剪接模式。

该研究加深了人们对于RNA高级结构与剪接调控之间关系的理解,首次证明了MYC调控蛋白FUBP1也同样具有调控剪接反应的活性,这种剪接调控能力很可能与FUBP1在多种重要生理过程中发挥的功能相关。

该项目得到了中科院、国家自然科学基金委、科技部及上海市科委项目的支持。(生物谷Bioon.com)

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression

Huang Lia, Zhijia Wanga, Xuexia Zhoua, Yuanming Chenga, Zhiqin Xiea, James L. Manleyb,1, and Ying Fenga,c,1

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3′splice site, and the other involves an exonic splicing silencer (ESS) situated 3′ to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.

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