title: A study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa (ADRP).
abstract: In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin beta1 for translocation to the nucleus, with no requirement for importin alpha1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with tau approximately 6 s and a slow component with tau approximately 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients.
authors.affiliation: Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Wilkie
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Morris
authors.forename: Shomi S
authors.initials: SS
authors.lastname: Bhattacharya
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
authors.forename: David M
authors.initials: DM
authors.lastname: Hunt
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D009154:Mutation; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
chemical_list: D005136:Eye Proteins; C424166:KPNB1 protein, human; C454222:PRPF31 protein, human; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C087978:karyopherin alpha 2