Jeffrey Smith, Advanced Technology & Biology division

Jeffrey Smith, Advanced Technology & Biology division

Location: 
Online
Start Time: 
Wed, 02/12/2020 - 1:00pm
End Time: 
Wed, 02/12/2020 - 2:00pm

WEHI Wednesday Seminar hosted by Associate Professor Andrew Webb

Jeffrey Smith, PhD Student, Webb Laboratory, Advanced Technology & Biology division - New Medicines & Advanced Technologies Theme

This is a PhD completion seminar

The Capture and Identification of Proteins That Bind RNA

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Includes Q&A session

 

RNA-binding proteins (RBPs) are typically regarded as facilitators of gene expression. In recent years, however, RNA-protein interactions have also emerged as a pervasive force in the regulation of homeostasis. The compendium of proteins with provable RNA-binding function has swollen from the hundreds to the thousands astride the partnership of MS-based proteomics and RNA sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that extract protein crosslinked to RNA in native conditions. These methods reveal snapshots in time displaying an extensive network of regulation and a provide wealth of data that can be used to discovery RNA-binding activity and the molecular interfaces at which it occurs. Though powerful, these methods remain limited in their range of experimental applications and identification often depends on destruction of the RBP-RNA complex.

Here I describe a new method developed to purify such complexes fully intact and from very low levels of transcriptomic input. This method differs from other RBP-discovery protocols in that it purifies complexes so completely that RBP identification can be conducted qualitatively and without differential abundance analysis. This extraction method leaves protein and the bound RNA transcript intact It also permits target-specific and sequence agnostic captures.

The performance of this method is first assessed with a census of proteins that directly interact with global, or targeted, RNA transcripts from model cell lines. The broader sensitivity and application of the method is then extended to investigate how protein-RNA interactions change during the transition from quiescence to proliferation then contraction in primary murine CD8+ T cells. These studies demonstrate how cellular responses provoke different proteins to moonlight as RNA binders and sheds light on a network of complex, co-evolved molecular machines.