Poster abstracts

Poster number 20 submitted by Andrew Giltmier

Identifying novel protein components of the cytoplasmic capping complex using proximity-dependent biotinylation

Andrew J. Giltmier (Biological Chemistry and Pharmacology), Jackson B. Trotman (Biological Chemistry and Pharmacology)

Abstract:
mRNAs that appear without a 5’ cap in the cytoplasm are readily identified and degraded by cytoplasmic enzymes. However, a smaller set of uncapped mRNAs can be recapped in the cytoplasm, protecting them from degradation by exonucleases, allowing them to return to a translationally active state [1]. The cytoplasmic capping complex consists of cytoplasmic capping enzyme (cCE), RNA-guanine-7methyltransferase (RNMT), and a 5’ RNA kinase that assemble on adapter protein Nck1. This complex is the set of proteins responsible for replacing the 5’ cap that provides a unique form of mRNA regulation [2]. Although the basic structure of the cytoplasmic capping complex is established, many details remain unknown. Our goal is to complete our understanding of the structure of this complex using a new in vivo approach for proximity-dependent labeling of proteins called BioID (proximity-dependent biotin identification). Using a promiscuous biotin ligase bound to cCE, we identify known and unknown candidates of the cytoplasmic capping complex by biotin tagging proteins as they interact with the complex. These tagged candidate proteins are isolated via streptavidin affinity chromatography and identified with mass spectrometry analysis. Identification of unknown interactors has the potential to better understand the biological relevance of a unique mechanism of post-transcriptional regulation.

References:
Otsuka Y, Kedersha NL, Schoenberg DR. Identification of a cytoplasmic complex that adds a cap onto 5′-monophosphate RNA. Mol Cell Biol. 2009; 29: 2155–2167

Mukherjee C., Bakthavachalu B., Schoenberg DR. The cytoplasmic capping complex assembles on adapter protein Nck1 bound to the proline-rich C-terminus of mammalian capping enzyme. PLoS Biol. 2014; 12:E1001933.

Trotman JB, Mukherjee C, Giltmier AJ, Schoenberg DR. Identification of RNMT as a component of the cytoplasmic capping complex. Submitted (Moleular Cell). 2017.

Roux KJ, Kim DI, Raida M, Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012; 196:801–10.

Keywords: Capping , BioID