Disruption of PRPF31 leads to deregulated expression and altered splicing of genes involved in phototransduction

Purpose: PRPF31 as a general mRNA splicing factor has important roles in every cell of each organ in the body. But, there is not enough evidence to clarify how PRPF31 deficiency is associated with retina specific disease as Retinitis Pigmentosa (RP). In this study, we investigated the functional effects of PRPF31 downregulation in a model of Human Organotypic Retinal Culture (HORC) using RNA sequencing technique.Methods: Human eyes from young donors (20-40 years old) were obtained within the 24 hours post mortem from Iranian eye bank. The retinas were isolated and transfected using PRPF31 and scramble siRNA. PRPF31 downregulation was confirmed by quantitative real time PCR (q-RTPCR). The mRNA libraries were sequenced on Illumina HiSeq 4000 and analyzed using bioinformatics approaches. Then, q-RTPCR for some downregulated genes was performed for additional confirmation.Results: PRPF31 reduction in siRNA treated samples led to downregulation of 1040 of genes and upregulation of 858 of genes, as a whole. The differentially downregulated genes were enriched in phototransduction (RHO, ROM1, FSCN, GNAT, CRX, NR2E3) and rhodopsin mediated visual cycle. RNA sequencing analysis confirmed q-RTPCR results of selected genes involved in phototransduction. In addition, genes with increased expression were mainly associated with immune and inflammatory responses, particularly innate immune system. Also, we examined the effects of PRPF31 deficiency on splicing by analysis of differential exon usage (DEU) of the retina. Pathway analysis of DEU genes indicated that the most affected pathways are the phototransduction, cortical actin cytoskeleton organization and photoreceptor cell development .Conclusion: Our data reveals that PRPF31 deficiency has a crucial role in retina specific pathology by affecting the expression and exon usage of wide range of genes implicated in phototransduction and rhodopsin mediated signaling pathway. In addition, there was an evidence of upregulation of some genes which bioinformatics approach showed their important role in activation of inflammatory response. Together these findings not only confirm our model of RP11 but also unravel some mysteries in pathophysiology of this disease in human retina

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