Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Global and sequence-targeted purification of intact RBP-RNA complexes (#83)

Jeffrey Smith 1 2 , Stephen Wilcox 1 , Laura Dagley 1 , Jarrod Sandow 1 , Rune Larsen 1 2 , Aaron Jex 1 , Melissa Davis 1 , Andrew Webb 1
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. The Department of Medical Biology, The University of Melbourne, Melbourne, VIC, Australia

The lifespan of an RNA molecule from synthesis to degradation, and transcription to translation, is regulated by RNA binding proteins (RBPs). The identification and characterisation of RBPs must therefore define our first steps towards understanding how the post-transcriptional control of gene expression might impact homeostasis or disorder. Yet RNA Binding is a temporal phenomenon and, coupled with its potency of effect, represents a difficult model to study. Recent efforts to discover the corpus of an RNA-binding proteome have focused on the development of biochemical techniques to isolate protein-RNA complexes for mass-spectrometry (MS). To achieve this we developed a complete in-solution method to recover clean, purified RBP-RNA completely intact.  Moreover, the stringency of these purifications is enough to permit qualitative confirmation of RNA-binding activity; while the efficiency of extraction can support complex, label-free experiments targeting quantitative changes in dynamic RBP-activity. Finally, we share preliminary results that integrate RNA-hybridisation probes to target RNA species based on common sequence elements and thence identify their protein binding partners.