Combinatorial mRNA Regulation by miRNAs and Pumilio proteins [CLIP-seq]

Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3′ untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3′ UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3′ UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent. Determination of AGO2 binding by CLIP-seq in T-REx-293 cells with and without PUM1 and PUM2 knockdown

约1500种RNA结合蛋白（RNA-binding proteins, RBPs）可通过调控特定转录本集合深刻影响哺乳动物细胞功能，这类蛋白通常通过与3′非翻译区（3′ untranslated regions, UTRs）的序列特异性结合，调控信使RNA（mRNA）的稳定性与翻译过程。除了各自独立的调控作用外，RNA结合蛋白在特定mRNA上形成的高阶组合相互作用，被认为是该调控网络的核心基础。为评估此类协同调控的覆盖范围，我们采用全局实验方法结合靶向验证策略，对两种已被充分表征且高度保守的RNA结合蛋白——Argonaute2（AGO2）与Pumilio（PUM1和PUM2）——的相互作用展开研究。通过CLIP测序（CLIP-seq）定量分析Pumilio基因敲低后，AGO2与mRNA结合的全转录组变化；结果显示，同一3′UTR上存在Pumilio结合位点的情况，与AGO2结合占有率的协同或拮抗效应显著相关。此外，与AGO2结合位点重叠的Pumilio结合位点，在AGO2结合被阻断后呈现出减弱且具有差异的结合特征，提示二者存在协同相互作用。在针对AGO2与Pumilio共定位的候选3′UTR位点开展的荧光素酶报告基因（luciferase reporter）验证实验中，我们鉴定出3个存在拮抗相互作用的位点：Pumilio可抵消微小RNA（microRNA, miRNA）介导的基因沉默效应。有趣的是，两种蛋白的结合位点间距过大，无法通过空间位阻产生拮抗作用，这提示存在另一套调控机制。本研究数据从实验层面验证了组合调控模型，并表明原本主要发挥抑制功能的Pumilio蛋白，可根据所处环境的不同改变其调控行为模式。综上，本研究方法凸显了进一步阐明RNA结合蛋白间复杂相互作用及其全转录组覆盖范围的重要性。本研究通过CLIP-seq测定了存在或不存在PUM1和PUM2基因敲低的T-REx-293细胞中AGO2的结合情况。