Item

Abstract

Abstract

Introduction: Mutations in pre-mRNA processing factor 31 (PRPF31), a core
protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant
retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiqui-
tously expressed tri-snRNP proteins result in retina-specific disorders, and so far,
the underlying mechanism of splicing factors-related RP is poorly understood.
Methods: We used the induced pluripotent stem cell (iPSC) technology to gen-
erate retinal organoids and RPE models from four patients with severe and very
severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic con-
trol.

Results: To fully assess the impacts of PRPF31 mutations, quantitative pro-
teomics analyses of retinal organoids and RPE cells were carried out showing
RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and
visual cycle-related pathways to be significantly affected. Strikingly, the patient-
derived RPE and retinal cells were characterised by the presence of large
amounts of cytoplasmic aggregates containing the mutant PRPF31 and mis-
folded, ubiquitin-conjugated proteins including key visual cycle and other RP-
linked tri-snRNP proteins, which accumulated progressively with time. The
mutant PRPF31 variant was not incorporated into splicing complexes, but reduc-
tion of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bod-
ies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and
reduced formation of active spliceosomes giving rise to global splicing dysregu-
lation. Moreover, the impaired waste disposal mechanisms further exacerbated
aggregate formation, and targeting these by activating the autophagy pathway
using Rapamycin reduced cytoplasmic aggregates, leading to improved cell sur-
vival.

Conclusions: Our data demonstrate that it is the progressive aggregate accumu-
lation that overburdens the waste disposal machinery rather than direct PRPF31-
initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplas-
mic aggregates presents a novel therapeutic strategy that can be combined with
gene therapy studies to fully restore RPE and retinal cell function in PRPF31-
adRP patients.