Abstract
Purpose :
Higher organisms have developed the ability to repurpose genes to have multiple functions by expressing numerous isoforms from the same gene locus. Expression of these isoforms can be tightly regulated so that individual cell types can express a subset of isoforms tailored to that particular cell’s needs. When disease causing genes have transcriptional patterns that fit this profile, it is increasingly difficult to pinpoint the biological mechanism that causes diseasebecause multiple cell types and isoforms are involved. Further, mutations that only affect select isoforms/cell types may display different phenotypes from mutations that affect all isoforms/cell types. The gene CRB1 is associated with various retinopathies of differing severity. Intriguingly, CRB1 is also known to express multiple isoforms. To date, a comprehensive evaluation of CRB1 isoforms and their contribution to retinal disease has yet to be performed.
Methods :
We developed a method for targeted long-read PacBio sequencing of Crb1 transcripts at multiple mouse retinal developmental timepoints. This allowed us to determine the full repertoire of Crb1 isoforms and their relative expression. Additionally, we performed the same sequencing on human CRB1 to determine whether isoforms and expression patterns are conserved across species. We mapped isoform expression in tissue using BaseScope in situ hybridization and verified protein expression with isoform specific antibodies. Finally, we generated two new mouse models of Crb1 to determine how the isoforms contribute to disease.
Results :
We discovered several novel isoforms of Crb1 that are abundantly expressed in the retina. Notably, we found a novel isoform that is unique to photoreceptors. The novel photoreceptor isoform is more abundant than the canonical isoform expressed by muller glia and has several unique protein domains that differ from the canonical form. Isoform expression in mouse is consistent with human expression and also conserved across vertebrates. The newly developed Crb1 mouse models display different phenotypes from one another and from existing models.
Conclusions :
Our results suggest that multiple isoforms and cell types contribute to the phenotypes of CRB1 retinopathies. This project provides a framework for determining how isoforms differentially contribute to gene function and disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.