Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Abstract

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.

Mutations in the Spliceosome Component prp-6 and Overexpression of cdh-5 Suppress Axon Guidance Defects of cdh-4 Mutants in Caenorhabditis elegans

“…Partial loss-of-function of prp-6 causes misregulation of several 100 genes, including the cadherin cdh-5 . 11 We found that overexpression of cdh-5 can partially suppress axonal defects in cdh-4 mutants, suggesting that cdh-5 can partially compensate for the loss of cdh-4 .…”

“…This confirms that the suppression of axonal defects is caused by a mutation in prp-6 . Complete loss-of-function of prp-6 is lethal, 11 since splicing is an essential cellular process. Therefore, prp-6(hd170) is likely to be a partial loss-of-function allele.…”

Splicing-related Retinitis Pigmentosa mutations mimicked in C. elegans allow the identification of disease modifiers and drug screens

“…We chose PRPF8 and SNRNP200 because these two proteins physically interact through the C-terminal region of PRPF8, where the s-adRP mutations are located (10). Moreover, RNAi of their orthologs in C. elegans, along with PRPF6, showed the most penetrant phenotypes (26). These three genes encode proteins of the U5 complex and, based on our previous RNAi experiments, would be more prone to harbor missense mutations with a strong phenotype than the other three splicing factors (PRP3, PRPF31 and PRPF4), which are part of the U4 complex.…”

“…Such extraordinary conservation would imply maintenance of the function and therefore the missense mutations could have similar consequences at the molecular level in humans and in nematodes. C. elegans has already been exploited as model for s-adRP (26) and would be further exploited in the future if functional replacement of C. elegans s-adRP genes at its endogenous locus can be achieved with their human counterparts. The challenge here would be to express a functional human splicing factor at endogenous levels without causing any phenotype in the nematode.…”

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

“…These seven genes are PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, SNRNP200 and RP9. All of them, except for RP9, encode highly conserved proteins between C. elegans and humans [114]. Photoreceptor cells are characterized by an intense transcriptional activity and metabolic rate [115] and, even though they are absent in C. elegans, the animal does present other cells that have similar increased metabolic rate and high transcriptional levels during the larval phase [116,117].…”