Mutant Prpf3 Pre–mRNA Splicing Factor Aggregates Inside the Nucleus of Neuronal Cells

Abstract

Purpose: : Different genes encoding pre–mRNA splicing factors (PRPF31, PRPF8, PRPF3 and PAP1) were found mutated in autosomal dominant RP. The link between mutant splicing factors and retinal degeneration is completely obscure. Analysis of the subcellular localization and interaction with other splicing factors in different cell lines will be fundamental in defining why mutation of PRPF3 causes RP and whether RP18 is due to a dominant effect of the mutant protein or haploinsufficiency.

Methods: : Co–localization studies by double immunofluorescence of the endogenous PRPF3 protein and other proteins that label different subnuclear compartments were undertaken. Mutant forms of PRPF3 (P493S and T494M) were transfected in different cell lines to define subcellular localization of mutant PRPF3 compared to wild type. Transfected cells were treated with drugs changing transcriptional or proteasomal activities in order to define whether metabolic changes affect mutant PRPF3 behavior in epithelial or neuronal cells.

Results: : We analyzed subcellular distribution of PRPF3 in HeLa cells by immunofluorescence and found co–localization with snRNPs and partial co–localization with splicing nuclear speckles. In normal conditions transfected mutant PRPF3 shows a subnuclear localization similar to the wild type. The mutant but not the wild type protein forms aggregates in HeLa cells when either transcriptional or proteasome activities are altered. A similar behavior of mutant PRPF3 was observed in in vitro differentiated neurons. We also observed that mutant PRPF3 caused apoptosis in these conditions.

Conclusions: : Expression studies failed to define a direct correlation of PRPF3 transcript distribution with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. We otherwise found that mutant PRPF3 shows a pathological behavior specifically in neurons. The abnormal formation of aggregates by mutant PRPF3 was correlated to specific metabolic changes in cells.

Keywords: retinitis • mutations • apoptosis/cell death