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Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants.
RNA Biol 2019; :1-10RB

Abstract

Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins.

Authors+Show Affiliations

Department of Life Sciences and Biotechnology, University of Ferrara , Ferrara , Italy.Department of Life Sciences and Biotechnology, University of Ferrara , Ferrara , Italy.Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara , Ferrara , Italy.Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara , Ferrara , Italy.Department of Life Sciences and Biotechnology, University of Ferrara , Ferrara , Italy.Department of Life Sciences and Biotechnology, University of Ferrara , Ferrara , Italy.Department of Life Sciences and Biotechnology, University of Ferrara , Ferrara , Italy.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31613176

Citation

Lombardi, Silvia, et al. "Translational Readthrough of GLA Nonsense Mutations Suggests Dominant-negative Effects Exerted By the Interaction of Wild-type and Missense Variants." RNA Biology, 2019, pp. 1-10.
Lombardi S, Ferrarese M, Marchi S, et al. Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants. RNA Biol. 2019.
Lombardi, S., Ferrarese, M., Marchi, S., Pinton, P., Pinotti, M., Bernardi, F., & Branchini, A. (2019). Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants. RNA Biology, pp. 1-10. doi:10.1080/15476286.2019.1676115.
Lombardi S, et al. Translational Readthrough of GLA Nonsense Mutations Suggests Dominant-negative Effects Exerted By the Interaction of Wild-type and Missense Variants. RNA Biol. 2019 Oct 15;1-10. PubMed PMID: 31613176.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants. AU - Lombardi,Silvia, AU - Ferrarese,Mattia, AU - Marchi,Saverio, AU - Pinton,Paolo, AU - Pinotti,Mirko, AU - Bernardi,Francesco, AU - Branchini,Alessio, Y1 - 2019/10/15/ PY - 2019/10/16/entrez KW - Nonsense mutations KW - fabry disease KW - lysosomal disorders KW - translational readthrough SP - 1 EP - 10 JF - RNA biology JO - RNA Biol N2 - Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins. SN - 1555-8584 UR - https://www.unboundmedicine.com/medline/citation/31613176/Translational_readthrough_of_GLA_nonsense_mutations_suggests_dominant-negative_effects_exerted_by_the_interaction_of_wild-type_and_missense_variants L2 - http://www.tandfonline.com/doi/full/10.1080/15476286.2019.1676115 DB - PRIME DP - Unbound Medicine ER -