Talk abstracts

Talk on Friday 02:00-02:15pm submitted by Chandrama Mukherjee

Adaptor protein Nck1 interacts with cytoplasmic capping enzyme and regulates cytoplasmic capping

Chandrama Mukherjee (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, Ohio State University), Daniel R Schoenberg (1Center for RNA Biology, Department of Molecular & Cellular Biochemistry, Ohio State University)

Abstract:
Background: The addition of 5’ cap is the first step in pre-mRNA processing and this reaction is catalyzed by the nuclear capping enzyme associated with RNA pol II. The cap plays a central role in subsequent steps of processing steps and in translation initiation, and loss of cap was thought to irreversibly commit mRNA to decay. In contrast to this notion, our lab identified a ~140 kDa cytoplasmic complex of capping enzyme together with a 5’-monophosphate RNA kinase that converts uncapped mono-phosphate RNAs to diphosphate substrates for mRNA capping enzyme. Cytoplasmic capping functions to control the cycling of the target mRNAs between translating and non-translating states, and plays a role in cellular recovery from stress as overexpression of inactive capping enzyme reduces the ability of cells to recover from oxidative stress.

Principal Finding: In the present study we show Nck1, a SH3/SH2 adaptor protein binds directly with cytoplasmic capping enzyme. The association is RNase independent and requires the third SH3 domain of Nck1. The proline rich sequence in both N-terminal and C-terminal domains of capping enzyme are potential sites for Nck1 binding, and we show that the C-terminal domain of capping enzyme (572-597) is required for Nck1 interaction as well as for cytoplasmic capping. The reconstitution of a functional complex following addition of recombinant Nck1 to a cytoplasmic extract prepared from cells expressing epitope tagged capping enzyme provided direct evidence of a role for Nck1 in assembly of the cytoplasmic capping enzyme complex. Finally, reduced activity of immunoprecipitated cytoplasmic capping enzyme complex from cells in which Nck1 was knocked down provided functional evidence of a role for Nck1 in cytoplasmic capping.

Conclusion: Our results identify a previously unknown interaction between Nck1 and cytoplasmic capping enzyme. We suggest Nck1 serves as the scaffold for the recruiting of the 5’-monophosphate RNA kinase to the cytoplasmic capping enzyme. Experiments are in progress to identify the 5’-monophosphate RNA kinase. Our study establishes that Nck1, an adapter protein known to mediate receptor tyrosine kinase signaling at the membrane level, also regulates gene expression through cytoplasmic capping.

Supported by grant R01 GM084177 from the National Institute of General Medical Sciences, National Institutes of Health.

Keywords: Cytoplasmic capping, Cytoplasmic capping enzyme, SH3 domain