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In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs

Lualdi, Susanna, Del Zotto, Genny, Zegarra-Moran, Olga, Pedemonte, Nicoletta, Corsolini, Fabio, Bruschi, Maurizio, Tomati, Valeria, Amico, Giulia, Candiano, Giovanni, Dardis, Andrea, Cooper, David Neil and Filocamo, Mirella 2017. In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs. Human Mutation 38 (7) , pp. 849-862. 10.1002/humu.23243

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Abstract

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct “edited” iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the “corrected” IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients’ cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Uncontrolled Keywords: confocal high-content analysis, confocal microscopy, edited IDS transcripts to wild-type, expression vectors, imaging flow cytometry, RNA editing conversion and deletion, SDS-PAGE, single nucleotide primer extension, variant correction, western blot
Publisher: Wiley-Blackwell
ISSN: 1059-7794
Date of First Compliant Deposit: 19 June 2017
Date of Acceptance: 22 April 2017
Last Modified: 08 May 2018 21:44
URI: http://orca.cf.ac.uk/id/eprint/101546

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