Supplementary MaterialsSupplementary Information 41467_2020_17009_MOESM1_ESM
April 7, 2021
Supplementary MaterialsSupplementary Information 41467_2020_17009_MOESM1_ESM. (individual isoform is the only one indicated by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we determine a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Consequently, our isoform recognition strategy enables finding of fresh gene functions relevant to disease. gene13, BAY-8002 the mammalian clustered protocadherins14, and the neurexin gene family15,16. Each of these genes produces hundreds of protein isoforms with unique binding specificity, diversifying the molecular acknowledgement events that mediate assembly of the nervous system17C19. From these good examples it seems obvious that, to understand the molecular basis for neural circuit wiring, it will be necessary to define the precise repertoire of cell-surface protein isoforms expressed in the developing CNS. A second reason for focusing on cell-surface molecules is that genetic alterations influencing them have been implicated in numerous CNS disorders. These include autism20, epilepsy21,22, and BAY-8002 neurodegeneration23C26. However, in the vast majority of these instances, it remains unclear why particular mutations increase disease risk. Comprehensive isoform identification offers great potential to reveal how these genetic variants cause disease pathology. Here we devised a strategy that leverages Pacific Biosciences (PacBio) long-read sequencing technology to generate comprehensive catalogs of CNS cell-surface molecules. Long-read sequencing is ideal for full-length transcript recognition; however, sequencing depth is not yet adequate to reveal the full scope of isoform diversity27C30. To conquer this limitation we adapted a strategy from short-read sequencing, in which targeted cDNAs GPSA are drawn down with biotinylated probes against known exons31,32. This approach yielded major improvements in long-read protection, exposing an rich diversity of isoforms encoded with the targeted genes unexpectedly. To make feeling of these complicated datasets, we created bioinformatics equipment for the evaluation and classification of isoforms, and for identifying their appearance patterns using short-read RNA-seq data. To show how our strategy can illuminate gene function, we examined one gene, is normally an associate from the conserved Crumbs gene family members, which encode cell-surface proteins that mediate apico-basal epithelial polarity33. Within the retina, CRB1 localizes towards the outer limiting membrane (OLM), a set of structurally important junctions between photoreceptors and neighboring glial cells known as Mller glia26. OLM junctions form at exact subcellular domains within each cell type, suggesting a high degree of molecular specificity in the establishment of these intercellular contacts34. There is great desire for understanding the function of CRB1 at OLM junctions, because loss-of-function mutations in human being cause a spectrum of retinal degenerative disorders35. It has been proposed that loss of OLM integrity might play a role in disease pathogenesis26,36, but studies in mice have yet to convincingly support this model: Deletion of the known isoform neither disrupts the OLM nor causes significant photoreceptor degeneration37. Here we determine a new isoform that is far more abundantin both mouse and human being retinathan the canonical isoform. Using mutant mice, we display that this isoform is required for OLM integrity and that its removal is required to properly phenocopy the human being degenerative disease. These results call for a major revision to prevailing models of CRB1 disease genetics and pathobiology. Thus, our findings provide a stunning example of how comprehensive isoform characterization can unveil important gene functions that were previously overlooked, enabling fresh insights into many biological questions including the biology of disease-associated genes. Results Cataloging isoforms via long-read BAY-8002 capture sequencing To define the isoform diversity of CNS cell surface molecules, we 1st by hand screened RNA-seq data from mouse retina and mind38,39 to identify genes that showed unannotated mRNA diversity. We focused on cell surface receptors of the epidermal growth element (EGF), Immunoglobulin (Ig), and adhesion G-protein coupled receptor superfamilies, as these genes have known tasks in cell-cell acknowledgement. For each gene screened (gene.