The regulation of cytokines is of vital importance for achieving homeostasis following the induction of the innate immune response; an inability to do so promotes the development of chronic conditions such as auto-immunity and cancer. Tristetraprolin (TTP) regulates the translation and stability of pro-inflammatory cytokine mRNAs containing AU-rich elements (AREs) through the recruitment of components involved in translation repression, deadenylation, decapping, and exonucleolytic degradation. Recently, the C-terminus of TTP has been shown to be a docking site for the CCR4-NOT deadenylase complex through its association with the scaffold protein CNOT1. This CNOT1-interacting motif (CIM) of TTP is highly conserved among TTP family members and is a site of phosphorylation; however, the relevance of this phosphorylation event in TTP regulation is currently unknown. Deadenylation is often regarded as the rate-limiting step of mRNA decay, suggesting that regulation of the TTP CIM is a critical checkpoint of the innate immune response. The objective of this research is to i) understand the effect that cell signaling events, acting on the CIM, have on the inflammatory response, and ii) investigate the consequence that mutating the CIM will have on target mRNA stability. Furthermore, although it is generally accepted that the removal of the poly(A) tail is sufficient to promote further mRNA decay, preliminary work suggest that despite little degradation of a reporter transcript, the TTP CIM is capable of activating deadenylation. It is possible that for TTP-mediated mRNA degradation to occur, deadenylation must be coupled with other decay-promoting events. Therefore, an additional goal of this project is to uncover TTP- domains that, when coupled with the CIM, promote the decay of target transcripts. In order to address these questions, a combination of molecular biology, human-based cell culture, mass-spectrometry, and next- generation sequencing techniques will be performed. The work proposed here offers insight into both the regulation of the inflammatory response in addition to offering insight into the mechanisms involving mRNA decay.
(Public Health Relevance Statement): A misregulated inflammatory response contributes to a variety of chronic pathologies including auto-immunity and cancer. Central to the proper induction and resolution of the inflammatory response is the stability and regulation of mRNA transcripts encoding pro-inflammatory cytokines. Investigating the cellular signaling events involved in regulating the RNA destabilizing factor tristetraprolin offers insight into gene expression changes during the innate immune response.