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

3UTRome 的整体变化切换对生长因子的反应

• 批准号: 9245281
• 负责人: Marco Mangone
• 金额: $ 19.87万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-10 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245281
• 关键词: 3' Untranslated Regions; Address; Adopted; CRISPR/Cas technology; Caenorhabditis elegans; Catalogs; Cells; Congenital Abnormality; Data Set; Development; Disease; Distal; EGF gene; Epidermal Growth Factor Receptor; Etiology; Event; Extracellular Signal Regulated Kinases; Family; Future; Gene Expression; Generations; Genes; Genetic Translation; Genome engineering; Global Change; Growth Factor; Hallmark Cell; Immunoprecipitation; Lead; Length; MEKs; Malignant Neoplasms; Mammalian Cell; Mediating; Messenger RNA; MicroRNAs; Microarray Analysis; Modality; PIK3CG gene; Pattern; Polyadenylation; Predisposition; Process; Protein Family; RNA-Binding Proteins; Ras/Raf; Regulation; Reporter; Repression; Research; Role; Signal Transduction; Source; Specialized Epithelial Cell; Surveys; System; Testing; Time; Tissues; Untranslated Regions; Variant; Vulva; Work; epigenome; genome editing; mRNA Stability; notch protein; precursor cell; prevent; programs; protein E; response; transcriptome; tumorigenesis

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.

概括 RNA 结合蛋白如 Pumilio 家族 (PUF) 对 3'UTR 施加抑制，因此 mRNA 稳定性和翻译。替代性多聚腺苷酸化 (APA) 会导致 3'UTR 长度发生变化。 通过 APA 协调大规模缩短 3'UTR 可导致逃避 3'UTR- 介导的抑制信号，并且可能是发育过程中的关键调节机制。 发现了 3'UTR 介导的 PUF 抑制和 APA 逃避抑制的病例 因此，我们对这些相互作用的基因对之间的理解存在差距。 互动是在 3'UTRome 和 APA 的全球层面上精心策划的。 EGF 模式化了秀丽隐杆线虫外阴的 6 个等能外阴前体细胞 (VPC)，即 3 个。 最靠近 EGF 源的部分被诱导形成外阴，而远端的三个仍然保留 值得注意的是，该事件的保真度为 99.8%。 在未诱导的细胞中特异性表达，表明远端 VPC 产生 PUF- 和 3'UTR 依赖性程序对信号无反应，在种系中也是如此。 PUF 抑制 ERK/MAP 激酶；无反应的细胞也可能采用同样的机制。 一种 PUF 的种系免疫沉淀 (IP) 鉴定出许多潜在的目标 3'UTR。 从这个数据集中，我们从每个外阴信号传导的基因中识别出多个目标 mRNA 级联（EGFR→Ras→Raf→MEK→ERK、Notch→CSL、PI3K→PDK→Akt 和 EGFR→Ras→ RalGEF→Ral），我们勇敢地承认冗余的 PUF 共同抑制了所有四种基因的 mRNA。 确定了信号级联以区分信号无反应细胞和信号反应细胞。 我们的中心假设是从近端（短 3'UTR）切换到远端（长 3'UTR） 多腺苷酸化序列 (PAS) 的使用控制着从信号响应性到非信号响应性的切换 我们将通过自下而上和自上而下的结合来系统地检验这一假设。 我们建议：1) 测试外阴信号基因 PUF IP 列表中的 3'UTR。 在无反应细胞中介导 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) 验证选定的候选者 通过 CRISPR/Cas9 基因组编辑改变内源 3'UTR 和聚腺苷酸化信号， 并删除相关的 PUF，我们将通过以下方式确定对发展保真度的贡献。 APA，以及 APA 引起的 3'UTR 中抑制性接入点的变化。