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ITALIA DI LIEGRO

Kinetics of rat CSD-C2 binding to H3.3 RNA

  • Authors: Saladino, P.; Gygax, D.; Spies, P.; Schiera, G.; Di Liegro, I.; Di Liegro, C.
  • Publication year: 2017
  • Type: Articolo in rivista (Articolo in rivista)
  • OA Link: http://hdl.handle.net/10447/249034

Abstract

Cold-shock domain containing protein C2 (CSD-C2; also known as PIPPin) is an RNA-binding protein conserved in the evolution that interacts with the 3’-untranslated region (3’-UTR) of rat H1.0 and H3.3 histone messengers. Biolayer interferometry (BLI) is a technique that measures changes in an interference pattern generated from visible light, reflected from an optical layer, and a biolayer which contains molecules of interest. In this study, we used the BLI methodology in order to analyze and describe the binding properties of CSD-C2 and the mRNA encoding the rat brain histone protein H3.3. Recombinant CSD-C2 was incubated with in vitro transcribed, and biotinylated H3.3 RNA fragments bound to streptavidin-conjugated Octet optical biosensors. In order to define the RNA region involved in binding, we used RNA probes corresponding to different portions of H3.3 RNA 3’-UTR. In this study, we showed that CSD-C2 binds to the last 199 nucleotides of the H3.3 RNA 3’-UTR, and that the apparent affinity constant of the interaction is in the nanomolar range. In addition, this study confirmed that BLI can be a very efficient and reliable method for studying RNA-protein interactions.