SQUINT RNA BINDING PROTEINS IN THE ZEBRAFISH EMBRYO: AN APTAMER BASED RNA AFFINITY PURIFICATION.

Abstract

Nodal factors are members of the TGFß superfamily of secreted growth factors and behave as morphogens. In Zebrafish there are 3 Nodal-related proteins namely the Cyclops, Squint and Southpaw. Our lab showed that Squint RNA is maternally deposited in the early Zebrafish embryo and localizes asymmetrically to 2 cells of the 4 cell stage embryo. This localization is dependent on microtubules and the 3¿UTR of the RNA. In order to identify the proteins that bind to form the RNP complex I have used a Tobramycin RNA-aptamer-based affinity purification method. I fused the Tobramycin aptamer to Squint Untranslated Region (UTR) sequences or Globin UTR as controls and then use synthetic RNA as bait to pull down proteins that bind to the RNA. The method has been optimized for this purpose and the preliminary results are to be analyzed by Gene Ontology analysis and validated.