Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

中文翻译：

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人类tRNA剪接核酸内切酶的装配缺陷导致桥小脑发育不全的前tRNA加工受损

人类转移RNA前体（pre-tRNA）的内含子通过与RNA激酶CLP1结合的tRNA剪切核酸内切酶TSEN切除。TSEN / CLP1的突变发生在脑小脑发育不全（PCH）的患者中，但是，它们在疾病中的作用尚不清楚。在这里，我们显示内含子切除是由四聚体TSEN催化的，该四聚体TSEN由非活性异二聚体组装而成，独立于CLP1。剪接位点识别涉及pre-tRNA的成熟域和反密码子-内含子碱基对。TSEN15-34异二聚体的2.1-Å分辨率X射线晶体结构和差示扫描荧光分析表明PCH突变引起热不稳定。尽管重组突变体TSEN中的核酸内切酶活性没有改变，我们观察到组装缺陷和降低的pre-tRNA裂解活性，导致PCH患者来源的成纤维细胞中的pre-tRNA池失衡。我们的工作定义了人类内含子切除的分子原理，并提供了TSEN稳定性调节可能有助于PCH表型的证据。