A Drosophila model to study retinitis pigmentosa pathology associated with mutations in the core splicing factor Prp8

Abstract

Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, an essential component of the spliceosome. Over 20 different RP-associated PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown. Here, we establish a Drosophila melanogaster model for RP13 by introducing the nine distinct RP mutations into the fly PRPF8 ortholog prp8 and express the mutant proteins in precise spatiotemporal patterns using the Gal4/UAS system. We show that all nine RP-Prp8 mutant proteins negatively impact developmental timing, albeit to a different extent, when expressed in the endocrine cells producing the primary insect moulting hormone. In the developing eye primordium, uncommitted epithelial precursors rather than differentiated photoreceptors appeared sensitive to Prp8 malfunction. Expression of the two most pathogenic variants, Prp8 S>F and Prp8 H>R , induced apoptosis causing alterations to the adult eye morphology. The affected tissue mounted stress and cytoprotective responses, while genetic programs underlying neuronal function were attenuated. Importantly, the penetrance and expressivity increased under prp8 heterozygosity. In contrast, blocking apoptosis alleviated cell loss but not the redox imbalance. Remarkably, the pathogenicity of the RP-Prp8 mutations in Drosophila correlates with the severity of clinical phenotypes in patients carrying the equivalent mutations, highlighting the suitability of the Drosophila model for in-depth functional studies of the mechanisms underlying RP13 etiology. This article has an associated First Person interview with the first author of the paper.

Ecd promotes U5 snRNP maturation and Prp8 stability

“…The following Drosophila melanogaster strains were used: w 1118 (BDSC; RRID:BDSC_3605 ), ecd 1 ( 32 ), ecd Δ (this study), w;; FRT2A (BDSC; RRID:BDSC_1997 ), w;; FRT82B (BDSC; RRID:BDSC_2035 ), w; nubbin-Gal4, UAS-myr-mRFP ( nub>mRFP , this study), w; escargot-Gal4, UAS-GFP, tub-Gal80 TS ( esg TS > , 33 ), w; nubbin-Gal4, UAS-myr-mRFP; tub-Gal80 TS ( nub TS >mRFP , this study), w;; ecd l( 3 )23 FRT2A ( 22 ), w;; ecd Δ FRT2A (recombined in this study), w; UAS-ecd RNAi ( 22 ), w; UAS-prp8 RNAi (VDRC; ID18565), w;; UAS-Prp8 wt ( 34 ) , w;; UAS-SmD3::3xHA (FlyORF; F003987), w;; UAS - SmD3::3xHA, UAS-ecd RNAi (recombined in this study) , w; nubbin-Gal4, UAS-myr-mRFP; UAS - SmD3::3xHA, tub-Gal80 TS (this study) , w; nubbin-Gal4, UAS-myr-mRFP; ecd 1 (this study), w;; UAS - SmD3::3xHA, UAS-Prp8 wt (recombined in this study), w;; UAS - SmD3::3xHA, UAS-ecd RNAi , UAS-Prp8 wt (recombined in this study), eyFLP; act>y + >Gal4, UAS-GFP; FRT82B, tub-Gal80 ( 35 ), eyFLP; act>y + >Gal4, UAS-GFP; tub-Gal80, FRT2A (this study), w; UAS-Ecd wt ; FRT82B (this study) , w; UAS-Myc::Ecd TripleA ; FRT82B (this study), w; UAS-Myc::Ecd Δ 34 ; FRT82B (this study), w; UAS-Ecd wt ; ecd Δ FRT2A (this study), w; UAS-Myc::Ecd TripleA ; ecd Δ FRT2A (this study), w; UAS-Myc::Ecd Δ 34 ; ecd Δ FRT2A (this study). Details on Drosophila strains are described in Supplementary Table S1 .…”

“…Samples were resolved on 10% or 12% SDS-PAGE. Candidate proteins were detected by immunoblotting with primary antibodies: rabbit anti-dPrp8-CTD (1:1000, 34 ), rat-anti Ecd N-term (1:1000, 22 ), mouse anti-Flag M2 (1:1000, Sigma-Aldrich Cat# F1804, RRID:AB_262044 ), rabbit anti-Myc (1:2000, Cell Signaling Technology Cat# 2272, RRID:AB_10692100 ), rabbit anti-HA (1:2000, Abcam Cat# ab9110, RRID:AB_307019 ), mouse anti-ATP5α (1:2000, Abcam Cat# ab14748, RRID:AB_301447 ), rabbit anti-RFP (1:1000, MBL Cat# PM005, RRID:AB_591279 ), rabbit anti-GFP (1:2000, Acris Cat# TP401, RRID:AB_10013661 ) followed by incubation with corresponding goat anti-mouse (1:5000, Jackson ImmunoResearch Labs Cat# 715-035-150, RRID:AB_2340770 ), rabbit (1:5000, Jackson ImmunoResearch Labs Cat# 711-035-152, RRID:AB_10015282 ) and rat (1:5000, Jackson ImmunoResearch Labs Cat# 712-035-153, RRID:AB_2340639 ) HRP-conjugated secondary antibodies. Chemiluminescent signal was captured using ImageQuant LAS4000 reader (GE Healthcare, RRID:SCR_014246 ).…”

Retinitis pigmentosa associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule neurons

“…The etiology of PRPF8 -linked RP, comprises nearly two dozen of heterozygous genetic variants (De Erkenez et al, 2002; McKie et al, 2001; Ruzickova and Stanek, 2017) with predominance of missense mutations clustering to very C-terminus of PRPF8 that is responsible for the SNRNP200 modulation (Mozaffari-Jovin et al, 2013). Functional screens demonstrated that the majority of aberrant PRPF8 proteins exhibited altered ability to interact with spliceosome cofactors in human cell culture and impacted development including eye development in fly (Malinova et al, 2017; Stankovic et al, 2020). The only exception was the PRPF8 p.Tyr2334Asn substitution.…”

“…The only exception was the PRPF8 p.Tyr2334Asn substitution. This variant displayed in in vitro assays selective disruption of splicing efficiency and affected strongly pupation in Drosophila, but in contrast to the other pathological PRPF8 variants the PRPF8-Y2334N protein was properly incorporated into small nuclear ribonucleoprotein particles (snRNPs), key components of the spliceosome (Malinova et al, 2017; Stankovic et al, 2020). In addition, several RP-linked mutations in PRPF8 target codons at the very 3’ end of the PRPF8 coding sequence causing shortening or aberrant prolongation of the PRPF8 C-terminus (De Erkenez et al, 2002; Martinez-Gimeno et al, 2003; McKie et al, 2001; Tiwari et al, 2016).…”

“…Functional screens demonstrated that the majority of aberrant PRPF8 proteins exhibited altered ability to interact with spliceosome cofactors in human cell culture and impacted development including eye development in fly (Malinova et al, 2017;Stankovic et al, 2020). The only exception was the PRPF8 p.Tyr2334Asn substitution.…”