Introduction: Alternative cleavage and Polyadenylation (APA) is a crucial process required to diversify transcriptomes by altering the untranslated region (UTR) of messenger RNA (mRNA). The retina contains an abundance of APA 3’UTRs, and specific APA events have been associated with age-related macular degeneration (AMD), the most common form of retinal blindness in the western world. In this study, we explored the role of APA in the degenerating mouse retina.
Methods: Mice (C57BL/6J) were exposed to photo-oxidative damage (PD), a model which mimics several AMD pathologies, using high-intensity light (100K lux) for 1, 3, and 5 days. Gene expression was profiled using the Visium Spatial Gene Expression platform (10X Genomics), and the APA sites were comprehensively mapped in each retinal layer. Differential APA usage in each gene was compared to controls using the 1) proximal usage percentage and 2) 3’UTR length which is calculated based on the distance from stop codon to each APA site weighted by expression.
Results: We discovered and visualised a seesaw regulatory pattern of APA, showing that the inferior and superior retina harbour differential APA usage at different PD stages, including changes in neurofilament light chain (Nefl). Nefl is a neurodegenerative marker gene, which shows high proximal usage in the periphery of the superior retina following 1 day of PD. The preference in proximal usage seesaws to the inferior retina with 3 days of PD. Further, we show that the changes in 3’UTR length correlate with the biological changes during PD, including identification of a global shortening in mRNA related to vesicle transport, RAS signalling, endocytosis with increasing PD in the retinal pigmented epithelium (RPE).
Conclusion: This work identifies a proposed new mechanism of RNA modification involved in retinal degenerations, further demonstrating the use of spatial transcriptomics as a powerful technique for elucidating changes in neurodegeneration.