Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing

Abstract

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.N eurons form highly specific and complex patterns of synaptic connections that underlie all brain function (1-5). Such specific connections require trillions of chemically differentiated synapses whose identity may be shaped by interactions of specific pre-and postsynaptic signaling molecules, especially cell-adhesion molecules. The genome size is insufficient to encode such diversity, but a mixture of combinatorial expression patterns that pair different synaptic cell-adhesion molecules with each other and of distinct alternative splicing patterns that amplify the number of cell-adhesion molecules into a large number of isoforms may generate the number of transsynaptic interactions needed to account for the enormous diversity of synaptic connections. In Drosophila melanogaster, alternative splicing of the mRNAs encoding the Down syndrome cell-adhesion molecule can generate nearly 20,000 protein isoforms whose structures specify axon bundling but not synapse formation (6). In mammals, alternative splicing of neurexin and some protocadherin mRNAs can also produce thousands of isoforms, which may at least in the case of neurexins be involved in synapse formation (7-10).Neurexins are type I membrane proteins that were discovered as presynaptic receptors for α-latrotoxin (8, 9). Six prin...

Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

“…Our RNA-Seq analysis detected and quantified all these previously described AS events, as well as many additional ones ( Fig. 1 C and D), including an alternative exon denoted AS6 that was observed recently (15). Unexpectedly, we observed a third promoter, which we refer to as γ promoter, in a highly conserved region between exons 23 and 24 that would generate a truncated isoform lacking almost all extracellular domains (Fig.…”

“…Three members of the family have been studied in detail and are estimated to generate over 3,000 variants through the use of two alternative promoters (α and β isoforms) and extensive AS in each family member (15) (Fig. 1 C-E and SI Appendix, Figs.…”

Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion

“…These data suggest that the interaction of Slitrks with LAR-RPTPs may be involved in initial synaptic specification rather than late synaptic maturation. The significance of alternative splicing in the transsynaptic adhesion pathway has been extensively explored in the case of the neurexin/neuroligin complex 1,30,31 . Similar to the case of neurexins, the splicing inserts of LAR-RPTPs alter its binding affinity to various postsynaptic partners, such as TrkC, IL-1RAcP and IL1RAPL1 (refs 2,3).…”

Recurrently Breaking Genes in Neural Progenitors: Potential Roles of DNA Breaks in Neuronal Function, Degeneration and Cancer

“…A number of RDC genes, for example, the neurexins (Treutlein et al 2014), are thought to produce numerous isoforms via differential RNA processing. Beyond such a diversification mechanism, we propose that RDC-based recombination, by generating exon deletions, might "hard-wire" expression of variant RDC products in NSPCs and, thereby, contribute to neural diversity.…”