Abstract
Purpose :
Neuronal transcriptomes are enriched with genes that express an abundance of mRNA isoforms. Generation of these isoforms, via alternative splicing and promoter use, expands the functional capacity of the genome, allowing one gene to encode multiple functionally distinct proteins. In neurons this facilitates the complex task of cell-cell recognition which orchestrates circuit development and function. Most transcriptomes are based on short-read RNAseq data, which is poorly suited to this purpose. Therefore, a large portion of the transcriptome is missing, hindering assessment of genes in regards to neural development, function and disease. To remedy this, we developed a new approach to survey mRNA diversity using long-read, single molecule sequencing.
Methods :
Retina RNAseq datasets from mouse were screened to identify genes with uncharacterized transcript diversity. Candidate genes were targeted for sequence capture and subsequent sequencing on the PacBio platform. Multiple mouse developmental timepoints were sequenced to identify temporal changes in isoform expression. RNAseq data and qRT-PCR were used to validate isoform expression and BaseScope in situ hybridization was used to map spatial expression of isoforms in retinal tissue. A proteogenomic approach was used to validate isoforms by mass spectrometry.
Results :
We found extensive isoform diversity beyond current transcriptome annotations. Notably, this method identified novel isoforms of the gene Crb1, an important retinal disease gene. These new isoforms are more abundant in retina than the isoforms that have been most frequently studied. Further, we found that Crb1 isoforms are temporally regulated in their expression onset and spatially segregated to differing retinal cell types. Sequence analysis predicts the isoforms will encode functionally distinct proteins. Profiling of human retina mRNA confirmed their existence in human.
Conclusions :
Across genes, isoform diversity exceeds current genome annotations. CRB1 is linked to several retinal degenerative disorders with dramatic phenotypic variability; the novel isoforms of CRB1 may transform our understanding of how the gene contributes to disease. Understanding CRB1 transcript diversity, expression patterns and function will be critical both to uncovering the mechanisms by which this gene contributes to diverse retinopathies, and also for developing effective treatments.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.