Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression
Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression
复制标题
DOI：
10.1073/pnas.1310607110
复制
发表时间：
2013-07-16
影响因子：
11.1
通讯作者：
Feng, Ying
中科院分区：
文献类型：
--
作者：
Li, Huang;Wang, Zhijia;Feng, Ying
mRNA前体的剪接由两个步骤组成，这两个步骤几乎总是紧密偶联以促进剪接mRNA的有效产生。然而，我们先前描述了在第一步之后完全封闭的剪接底物。我们现在已经研究了这种不寻常的第二步抑制的基础，并出乎意料地阐明了两个独立的机制。一个涉及位于3'剪接位点下游的茎环结构，另一个涉及位于该结构3'的外显子剪接沉默子（ESS）。这两个元件都有助于体外第二步阻断，也导致体内外显子跳跃。重要的是，我们确定了远上游元件结合蛋白1（FUBP 1），一个单链DNA和RNA结合蛋白，以前没有参与剪接，作为一个强大的ESS结合蛋白，和几个试验牵连它在ESS功能。我们证明了使用消耗/添加回实验，FUBP 1作为第二步阻遏物在体外和siRNA介导的敲低和过表达试验表明，它在体内调节外显子包含。总之，我们的研究结果提供了额外的见解剪接控制，并确定FUBP 1作为剪接调节器。
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.