The binding specificity and regulatory effect of WT and redesigned Puf2p [RNA-Seq]. Saccharomyces cerevisiae

PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3’UTRs, such that the protein tracks with its sites and binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network. Overall design: CLIP-seq was performed in BY4742 S. cerevisiae grown in log phase, and using 2 replicates of TAP-tagged proteins. RNA-seq was performed to determine the regulatory effect of WT or mutant Puf2p, using 4 replicates of the control (no Puf2p), 3 of WT Puf2p and 4 of R1 SNE Puf2p.

PUF蛋白（PUF proteins）已成为设计RNA结合蛋白以按需靶向结合并精准调控RNA的主流支架平台。本研究首次在体内全转录组层面解析了这类工程化改造的效应。通过HITS-CLIP与PAR-CLIP实验，我们证实酿酒酵母（S. cerevisiae）的非经典PUF蛋白Puf2p可结合超过1000个mRNA靶标。与经典PUF蛋白不同，Puf2p能够识别并结合多个UAAU基序。本研究还针对Puf2p的截短体开展了CLIP-seq实验，结果显示其朊蛋白结构域对于野生型（WT）的结合活性并非必需。我们设计了一款改造后的Puf2p，使其结合特异性从UAAU转向UAAG，由此实现蛋白与结合位点的精准匹配。在体内环境中，经重新设计的蛋白可特异性结合UAAG位点；其改变的结合特异性使蛋白从3'非翻译区（3'UTRs）发生重新分布，最终实现蛋白与其结合位点共定位，并在mRNA全长范围内进行结合。我们通过RNA-seq实验证实，R1 SNE Puf2p可抑制一套全新的RNA调控网络。实验整体设计如下：在对数生长期培养的BY4742酿酒酵母（S. cerevisiae）中开展CLIP-seq实验，采用2次生物学重复的串联亲和纯化（TAP）标记蛋白；为检测野生型或突变型Puf2p的调控效应，我们开展了RNA-seq实验，设置4次生物学重复的对照组（无Puf2p）、3次生物学重复的野生型Puf2p组，以及4次生物学重复的R1 SNE Puf2p组。