Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, CNRS, INSERM, PSL Research University, Paris, France.
Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Nat Commun. 2024 May 17;15(1):4209. doi: 10.1038/s41467-024-48371-5.
Exon junction complexes are deposited at exon-exon junctions during splicing. They are primarily known to activate non-sense mediated degradation of transcripts harbouring premature stop codons before the last intron. According to a popular model, exon-junction complexes accompany mRNAs to the cytoplasm where the first translating ribosome pushes them out. However, they are also removed by uncharacterized, translation-independent mechanisms. Little is known about kinetic and transcript specificity of these processes. Here we tag core subunits of exon-junction complexes with complementary split nanoluciferase fragments to obtain sensitive and quantitative assays for complex formation. Unexpectedly, exon-junction complexes form large stable mRNPs containing stalled ribosomes. Complex assembly and disassembly rates are determined after an arrest in transcription and/or translation. 85% of newly deposited exon-junction complexes are disassembled by a translation-dependent mechanism. However as this process is much faster than the translation-independent one, only 30% of the exon-junction complexes present in cells at steady state require translation for disassembly. Deep RNA sequencing shows a bias of exon-junction complex bound transcripts towards microtubule and centrosome coding ones and demonstrate that the lifetimes of exon-junction complexes are transcript-specific. This study provides a dynamic vision of exon-junction complexes and uncovers their unexpected stable association with ribosomes.
外显子连接复合物在剪接过程中沉积在外显子-外显子连接处。它们主要被认为能够激活含有过早终止密码子的转录本的无意义介导的降解，这些转录本位于最后一个内含子之前。根据一个流行的模型，外显子连接复合物与 mRNA 一起被运送到细胞质中，第一个翻译核糖体将它们推出。然而，它们也通过未被表征的、与翻译无关的机制被去除。关于这些过程的动力学和转录本特异性知之甚少。在这里，我们用互补分裂纳米荧光素酶片段标记外显子连接复合物的核心亚基，以获得用于复合物形成的敏感和定量测定。出乎意料的是，外显子连接复合物形成含有停滞核糖体的大稳定 mRNP。复合物的组装和拆卸速率是在转录和/或翻译被阻断后确定的。85%的新沉积的外显子连接复合物通过依赖翻译的机制被拆卸。然而，由于这个过程比非依赖翻译的过程快得多，只有 30%的稳定状态下存在于细胞中的外显子连接复合物需要翻译来进行拆卸。深度 RNA 测序显示外显子连接复合物结合的转录本偏向于微管和中心体编码的转录本，并证明外显子连接复合物的寿命是转录本特异性的。这项研究提供了外显子连接复合物的动态视角，并揭示了它们与核糖体的意外稳定关联。
