Department of Neurobiology, Duke University School of Medicine, Durham, NC, 27710, USA.
Department of Ophthalmology, Duke University School of Medicine, Durham, NC, 27710, USA.
Nat Commun. 2020 Jul 3;11(1):3328. doi: 10.1038/s41467-020-17009-7.
Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.
编码细胞表面蛋白的基因控制着神经系统的发育，并与神经紊乱相关。这些基因产生了仍未被充分描述的替代性 mRNA 异构体，阻碍了对疾病相关突变如何导致病理的理解。在这里，我们介绍了一种定义单个基因编码的全长异构体完整组合的策略。将这种方法应用于神经细胞表面分子，我们鉴定了数千种在视网膜和大脑中表达的未经注释的异构体。通过质谱分析，我们在体内证实了新发现的蛋白在细胞表面的表达。值得注意的是，我们发现了一个视网膜退化基因 CRB1 的主要异构体此前被忽视了。这种 CRB1 异构体是 CRB1 疾病中受影响的感光细胞唯一表达的异构体。利用小鼠突变体，我们确定了这种异构体在感光细胞-胶质细胞连接中的作用，并证明了这种异构体的缺失会加速感光细胞的死亡。因此，我们的异构体鉴定策略能够发现与疾病相关的新基因功能。
