Fiocco, F.; (2010) Identification of novel interacting partners of the pre-mRNA processing factor 31. Doctoral thesis , UCL (University College London).

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Abstract

Mutations in PRPF31 (RP11 locus) cause autosomal dominant retinitis pigmentosa (adRP), an inherited disorder of the retina characterised by degeneration of rod photoreceptors. PRPF31 is ubiquitously expressed and encodes a splicing factor involved in the spliceosome assembly. Interestingly, the phenotype caused by mutations in PRPF31 is restricted to the retina. This study aims to identify protein interactants of PRPF31, to contribute to a better understanding of the disease mechanism of RP11. Three potential interacting candidates were isolated through the yeast two-hybrid technique. Two of them (MAGI3, TRAK2) were confirmed to interact with PRPF31 using co-immunoprecipitation experiments after overexpression of the proteins; co-immunoprecipitation of the endogenous proteins was not carried out. MAGI3 and TRAK2 expression in human retina was proven by RT-PCR, however they are not retina-specific. MAGI3 and TRAK2 human retina transcripts did not appear to present major differences to the transcripts deposited in the Ensembl database. Tagged proteins were overexpressed in epithelial cells, and co-localisation between PRPF31 and each partner was mainly nuclear. In epithelial cells, both endogenous PRPF31 and MAGI3 localised to intercellular junctions but did not co-localise; however, after stress treatments, the proteins appeared to co-localise to the nucleus. In neuroblastoma cells, endogenous PRPF31 and TRAK2 did not co-localise. Immunohistochemistry experiments identified PRPF31 to be present largely in the inner segment and in the outer plexiform layer of murine retina. AdRP patients’ DNA was screened to detect mutations in MAGI3 that could be linked to retinal degeneration, but none was identified. The results are not conclusive of the molecular mechanism of RP11, but instead provide new grounds for research on PRPF31. These insights into the type of interacting partners and the localisations observed within the cell and the retina may cast new light into the function(s) of PRPF31, which may (or may not) link to retinal degeneration.

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