In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors

PLoS Genetics
Elvir BecirovicMartin Biel

Abstract

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results ...Continue Reading

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Citations

Aug 16, 2016·Frontiers in Neuroscience·Elvir BecirovicStylianos Michalakis
Oct 9, 2019·International Journal of Molecular Sciences·Frédéric Blond, Thierry Léveillard
Dec 10, 2019·Human Molecular Genetics·Lisa M RiedmayrElvir Becirovic
Apr 2, 2016·Human Molecular Genetics·O N Phuong NguyenElvir Becirovic
Aug 21, 2016·Cellular and Molecular Life Sciences : CMLS·Thierry Léveillard, José-Alain Sahel
Apr 2, 2020·Expert Opinion on Orphan Drugs·Moreno MenghiniRobert E MacLaren
Jun 20, 2020·Progress in Retinal and Eye Research·Nicole WeisschuhBernd Wissinger
Mar 5, 2018·Bio-protocol·Lisa Maria RiedmayrElvir Becirovic

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Methods Mentioned

BETA
fluorescence-activated cell sorting
PCR
FACS
transfection

Software Mentioned

ImageJ
Image Lab
ASSEDA
NNSplice
ASSEDA splice
LASAF

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