Any Way You Splice It: Analysis of Alternative Splicing in Macular, Nasal, and Temporal Retina as Assessed by RNA-Seq

Abstract

Purpose: We recently published a genome-wide analysis of differential expression between temporal, macular, and nasal regions of human neural retina and RPE/choroid. However, this analysis lumped all isoforms into a single expression index per gene. Given the vital role of alternative splicing and retinal-specific isoforms (e.g. MAK, ABCA4, SNRNP200, BBS8), we reanalyzed our dataset to evaluate regional differences at the transcript level.

Methods: 565 million paired RNA-Seq reads from temporal, macular, and nasal retina and RPE/choroid from four human donors (24 samples total) were mapped to human genome build hg19 using Tophat2, and transcript structures were identified using Cufflinks as detailed in (Whitmore et al. Exp Eye Res 2014). Differential expression was evaluated between regions within the same tissues (retina or RPE/choroid). Individual splicing isoforms were considered differentially expressed between regions if the expression was changed at least two-fold and the q-value < 0.01.

Results: When comparing regions within the retina, we found 67 and 148 differentially expressed transcripts in the macula-nasal and macula-temporal comparisons, respectively. DAVID analysis revealed enrichment for genes associated with locomotor activity and neurological signaling. Similar to our previously published gene-level analysis, no transcripts showed differential expression between nasal and temporal retina at our criteria. When comparing regions within the RPE/choroid samples, we found 42, 36 and 7 transcripts which were differentially expressed between macula and nasal, macula and temporal, and nasal and temporal, respectively.<br /> Intriguingly, close inspection of the mapped reads revealed a number of novel tissue-specific transcriptional events, such as an alternative transcription start site for CRB1 in retina.

Conclusions: While transcripts are differentially expressed between peripheral and macular regions of neural retina, we found no evidence of differential expression between nasal and temporal retina. This finding suggests that alternative splicing may not play a major role in retinal degenerations affecting the nasal and temporal regions to different degrees (e.g., MAK-associated retinitis pigmentosa). The identification of retina-specific exons expands our search space for disease-causing mutations within the exome.