Application Guide - QIAxcel Advanced

24 QIAxcel Advanced Application Guide 10/2016 Detection of alternative Tra2β regulated splicing Andrew Best, Sushma Grellscheid, and David J. Elliott, Institute of Genetic Medicine, Newcastle University, UK This application note describes how the QIAxcel® system was used to successfully determine the splicing pattern of exonic sequences targeted by Tra2β protein isoforms. Introduction Tra2β (Sfrs10) is an evolutionarily conserved splicing protein that is crucial for mouse embryogenesis (1), but its biological role is not fully understood. It has a modular structure with domains rich in arginine and serine (RS1 and RS2) and a central RNA recognition motif (RRM) that binds to target RNA sequences (2, 3). Furthermore, at least 3 isoforms of Tra2β have been identified. Tra2β is known to splice the Nasp histone chaperone gene, which monitors DNA double strand breaks (4). An evolutionarily conserved cassette exon (annotated Nasp-T) may play a crucial role in developmental processes. Tra2β splices Nasp via a number of binding sites, but the exact role of these interactions is not known. Because of the high levels of splicing inclusion observed for the wild type Nasp-T exon at endogenous cellular concentrations of Tra2β, we tested a mutated exon (“M3+M4”), which is less efficiently spliced, to find out whether the Tra2β binding sites are necessary for splicing activation. We also investigated the need for the Tra2β RRM and RS1 domains in these interactions (5). The QIAxcel system provides rapid, sensitive, and reproducible analyses of Tra2β regulated splicing. This system may also prove advantageous for studying the role of other splicing proteins and their target sequences. Materials and Methods HEK 293 cells were cotransfected with a mutated Nasp-T construct (M3+M4) and one of 3 Tra2β–GFP constructs encoding full length Tra2β, Tra2β∆RRM, or Tra2β∆RS1. Control cells were cotransfected with the Nasp-T construct (M3+M4) and GFP only.

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