Global changes in the 3'UTRome toggle responsiveness to growth factors

总结 RNA结合蛋白如Pumilio家族（PUF）对3 'UTR施加阻遏，因此 mRNA的稳定性和翻译。选择性多聚腺苷酸化（阿帕）导致3 'UTR长度变化。 通过阿帕协调大规模缩短3 'UTR可导致从3' UTR的逃避。 介导的抑制信号，并可能是一个关键的调控制度在发展过程中。然而大多数 发现了3 'UTR介导的PUF抑制和阿帕逃避抑制的确定病例 在相互作用的基因对之间。因此，我们对这些问题的理解存在差距。 在3 'UTRome和阿帕的全球水平上协调相互作用。 EGF在C.女阴秀丽线虫在6个等能的外阴前体细胞（VPC）中， 最接近EGF来源的被诱导形成外阴，而远端的三个仍然存在。 未诱导的值得注意的是，这一事件的发生具有99.8%的保真度。三个冗余PUF是 在未诱导的细胞中特异性表达，表明远端VPC制定了PUF-和 3 'UTR依赖性程序变得对信号无反应。在生殖细胞中， PUFs抑制ERK/MAP激酶;这种相同的机制可能被无反应性的细胞所采用。 VPC。一个PUF的种系免疫沉淀（IP）鉴定了许多潜在的靶3 'UTR。 从这个数据集中，我们从每个外阴信号传导中的基因中鉴定了多个靶mRNA。 级联（EGFR→Ras→Raf→MEK→ERK、Notch→CSL、PI 3 K →PDK→Akt和EGFR→Ras→ RalGEF→Ral）。我们假设多余的PUFs共同抑制所有四种基因的mRNA， 识别信号级联以区分信号无响应细胞和信号响应细胞。 我们的中心假设是从近端（短3 'UTR）到远端（长3' UTR）的转换 多聚腺苷酸化序列（PAS）的使用控制着从信号应答到非信号应答的转换。 响应能力。我们将结合自下而上和自上而下来系统地检验这一假设 明确的目标。我们建议：1）测试来自外阴信号传导基因的PUF IP列表的3 'UTR， 在无应答细胞中介导PUF依赖性报告基因抑制的能力，2）调查 VPC 3 'UTRome和全局近端到远端PAS切换，以及3）验证选择的候选项 通过CRISPR/Cas9基因组编辑改变内源性3 'UTR和多聚腺苷酸化信号， 以及删除相关联的PUF。我们将通过以下方式确定对发展忠诚度的贡献： 阿帕，以及阿帕引起的3 'UTR中抑制性通路点的变化。

SUMMARY RNA binding proteins like the Pumilio family (PUFs) exert repression on 3'UTRs, and hence mRNA stability and translation. Alternative polyadenylation (APA) causes 3'UTR length to vary. Coordinated large-scale shortening of 3'UTRs through APA can lead to evasion from 3'UTR- mediated repressive signals, and may be a key regulatory regime during development. Yet most identified cases of 3'UTR-mediated PUF repression - and APA to evade repression - were found ad hoc, between interacting gene pairs. Thus a gap exists in our understanding of how these interactions are orchestrated at the global level of the 3'UTRome and APA. EGF patterns the C. elegans vulva. Of six equipotent vulval precursor cells (VPCs), the three closest to the EGF source are induced to form the vulva, while the distal three remain uninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs are expressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and 3'UTR-dependent program to become non-responsive to signal. In the germline, the same PUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsive VPCs. Germline immunoprecipitation (IP) of one PUF identified many potential target 3'UTRs. From this dataset, we identified multiple target mRNAs from genes in each vulval signaling cascade (EGFR→Ras→Raf→MEK→ERK, Notch→CSL, PI3K→PDK→Akt, and EGFR→Ras→ RalGEF→Ral). We hypothesize that the redundant PUFs collectively repress mRNAs of all four identified signaling cascades to demarcate signal-non-responsive from signal-responsive cells. Our central hypothesis is that switching from proximal (short 3'UTR) to distal (long 3'UTR) polyadenylation sequence (PAS) usage governs switching from signal-responsiveness to non- responsiveness. We will systematically test this hypothesis by joining bottom-up and top-down specific aims. We propose to: 1) test 3'UTRs from the PUF IP list of vulval signaling genes for ability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey the VPC 3'UTRome and global proximal-to-distal PAS switching, and 3) validate select candidates by altering endogenous 3'UTRs and polyadenylation signals via CRISPR/Cas9 genome editing, and deleting associated PUFs. We will ascertain the contribution to developmental fidelity by APA, and the changes in repressive access points in the 3'UTRs caused by APA.