Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

Restricted oxygen supply from the choroid may lead to hypoxic conditions in the outer retina of the ageing eye and contribute not only to physiological aging but also to nonhereditary degenerative retinal diseases. To understand the hypoxic response of specific retinal cell types, we performed droplet-based single-cell RNA sequencing (scRNA-Seq) of retinas isolated from mice exposed to hypoxia. Except for ganglion cells, we identified all retinal cell types and a small RPE cluster among the 9410 normoxic and 9033 hypoxic cells in the analysis. Expression of hypoxic marker genes including Bnip3, Higd1a, and Egln1 was significantly upregulated in hypoxic clusters, confirming a general transcriptional response to hypoxia. Interestingly, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters and demonstrated isoform switching in hypoxia. The resulting short variants of these gene transcripts are connected to epitranscriptomic regulation, a notion supported by the differential expression of writers, readers and erasers of m 6 A RNA methylations in the hypoxic retina. By focusing on the hypoxic response in photoreceptors, we identified and confirmed a kinesin motor protein (Kif4) that was specifically and strongly induced in hypoxic cones. Retinal cells adapted to hypoxic conditions by adjusting their gene expression profile. Epitranscriptomic regulation of m 6 A RNA methylations may support the adaptational process. The cone-specific upregulation of Kif4, a motor protein with multiple functions, indicated a cone-specific response to hypoxia that may include cargo-delivery to specific intracellular locations, enrichment of transmembrane receptors or channels and/or modulation of chromatin structure. A male and a female wildtype mouse were exposed to 7% oxygen for 6 hours to induce hypoxic conditions in the retina. The retinas from hypoxic mice and a pair of male and female age-matched control mice were collected and dissociated for droplet-based single-cell RNA sequencing. The cell dissociates were fixed with methanol and the dissociates from the two animals under the same oxygen condition were combined. The two samples, "hypoxia" and "normoxia", were loaded onto separate lanes of a 10X Genomics chip and were prepared for sequencing using the v3 chemistry. The samples were then sequenced on an Illumina NovaSeq 6000.