Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Research Core, Research Facility Cluster, Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
EMBO J. 2020 Oct 15;39(20):e104708. doi: 10.15252/embj.2020104708. Epub 2020 Sep 14.
Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.
Let-7 是一种进化上保守的 microRNA，通过介导转录后基因沉默来调节广泛的生物学过程，包括发育、分化和肿瘤抑制。Let-7 的生物发生受到几种 RNA 结合蛋白的严格调控，包括 Lin28A/B，它抑制 let-7 的成熟。为了鉴定 Let-7 的新调控因子，我们设计了一种基于细胞的 RNA 结合蛋白功能筛选，使用 Let-7 传感器荧光素酶报告基因，并鉴定出 tRNA 假尿嘧啶合成酶 TruB1。TruB1 特异性增强了 let-7 家族成员的成熟。高通量测序交联免疫沉淀（HITS-CLIP）和生化分析表明，高吞吐量测序交联免疫沉淀（HITS-CLIP）和生化分析表明，内源性 TruB1 与 pri-let-7 的茎环结构直接结合，而不是诱导 miRNA 的假尿嘧啶化，Lin28A/B 也与 pri-let-7 的茎环结构结合。TruB1 选择性地增强了 pri-let-7 与介导 miRNA 成熟的 microprocessor DGCR8 之间的相互作用。最后，TruB1 抑制了细胞增殖，这部分是由 let-7 介导的。总之，我们揭示了 TruB1 在促进 let-7 成熟方面的一个意外功能。
