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Goranka Tanackovic, Adriana Ransijn, Philippe Thibault, Sherif Abou Elela, Roscoe Klinck, Eliot L. Berson, Benoit Chabot, Carlo Rivolta; Generalized Defects In Spliceosome Composition And Pre-mRNA Splicing Are Associated With Retinitis Pigmentosa In Humans. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1639.
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© ARVO (1962-2015); The Authors (2016-present)
Proteins PRPF31,PRPF8,and PRPF3(RP-PRPFs) are ubiquitously expressed components of the spliceosome, a complex processing nearly all pre-mRNAs. Although they are essential for survival of all cells, heterozygous mutations in human RP-PRPF genes cause nonsyndromic autosomal dominant retinitis pigmentosa (RP). The aim of this work is to study the role of PRPF mutations in the pathogenesis of RP and to assess the mechanisms by which systemic defects in splicing proteins cause disease only in the retina.
We used lymphoblastoid cells derived from 13 patients carrying 10 different mutations in all 3 RP-PRPF genes to study spliceosome composition, its assembly, and its function. In vitro functional tests were performed using nuclear extracts from cells. Endogenous RNA was studied by RT-PCR to assess splicing ex vivo. Finally, in silico approaches on microarray data were used to analyze the splicing process in various human tissues, including the retina.
We found that mutant RP-PRPF genes act through distinct molecular mechanisms, but all ultimately affect the stoichiometry of certain spliceosome components: U snRNAs (notably those present in the tri-snRNP) and some of the tri-snRNP proteins. We also observed that, in RP-PRPF extracts, the kinetics of spliceosome assembly are delayed at steps that are dependent on tri-snRNP functionality. All of these defects lead to deficiencies in both constitutive and alternative splicing, with at least 9% of endogenously expressed transcripts incorrectly spliced. The observed splicing deficiency is not mutation-specific, but rather depends on the RP-PRPF gene involved. Finally, we show that the steady-state levels of U snRNAs and spliced mRNAs are highest in the human retina.
Mutations in RP-PRPF genes can selectively affect splicing processes not only in the retina, but also in other tissues. We hypothesize that photoreceptor degeneration occurs in patients with RP-PRPF mutations because of the particularly elevated splicing needs of the retina.
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