PDF(Pubmed)
Abstract：
Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.
摘要：
伪尿苷(Φ),尿苷的异构体，普遍存在于RNA中，包括tRNA,rRNA,和mRNA。人假尿苷合酶3(PUS3)催化tRNA中位置38/39的假尿苷化。然而,它识别其RNA靶标并实现位点特异性的分子机制仍然难以捉摸。这里,我们确定了apo形式并与三个tRNA结合的PUS3的单颗粒冷冻EM结构，显示对称PUS3同二聚体如何识别tRNA并将靶尿苷定位在其活性位点附近。结构指导和患者来源的突变验证了我们在互补生化测定中的结构发现。此外，我们在HEK293细胞中删除了PUS1和PUS3，并通过Pseudo-seq定位了转录组范围的Φ位点。尽管在tRNA和mRNA中可以检测到PUS1依赖性位点，我们没有发现人类PUS3修饰mRNA的证据.我们的工作为人类PUS3介导的tRNA修饰提供了分子基础，并解释了其tRNA修饰活性如何与智力障碍有关。
