Departments of Bioengineering and Molecular and Cellular Physiology, School of Medicine, Department of Applied Physics, and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2014 Apr 1;111(13):E1291-9. doi: 10.1073/pnas.1403244111. Epub 2014 Mar 17.
Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.
神经连接蛋白是进化上保守的突触前细胞黏附分子，对于正常的突触形成和突触传递至关重要。间接证据表明，神经连接蛋白 mRNA 的广泛选择性剪接可能产生数百种（如果不是数千种）神经连接蛋白同工型，但目前还没有直接证据证明存在这种多样性。在这里，我们使用无偏倚的全长神经连接蛋白（Nrxn）1α、Nrxn1β、Nrxn2β、Nrxn3α 和 Nrxn3β mRNA 的长读测序，系统地评估了神经连接蛋白中使用了多少个具有显著频率的选择性剪接位点，以及这些位点的选择性剪接事件是否相互独立。在对超过 25000 条全长 mRNA 进行测序后，我们鉴定出了一种新的、大量使用的神经连接蛋白 1α 和 Nrxn3α 的选择性剪接外显子（称为选择性剪接序列 6），该外显子编码了在第五个 LNS（层粘连蛋白-α、神经连接蛋白、性激素结合球蛋白）结构域和第三个 EGF 样序列之间的柔性铰链区的 9 个残基插入。此外，我们还观察到了几个更大规模的选择性剪接事件，这些事件缺失了多个结构域，且比神经连接蛋白中典型的 6 个选择性剪接位点的频率要低得多。所有这 6 个典型的选择性剪接事件似乎都是相互独立的，这表明神经连接蛋白可能表现出比以前预期更大的同工型多样性，并包含数千种变体。我们的数据与以下观点一致，即α-神经连接蛋白代表细胞外蛋白相互作用支架，其中不同的 LNS 和 EGF 结构域介导不同的相互作用，这些相互作用影响不同的功能，并通过独立的选择性剪接事件独立调控。
