P120 The cytokine mRNA-interactome – An unbiased approach to identify RNA–protein interactions

Abstract

Introduction The first line of defense against invading pathogens is the innate immune system. Recognition of an intruder results in transcriptional up-regulation of several genes, including cytokines. The production of these potent immune mediators needs to be tightly monitored as aberrant cytokine expression results in tissue damage or development of autoimmune diseases. It becomes increasingly evident that post-transcriptional regulation of gene expression also strongly impacts on cytokine production, especially during the resolution phase of the immune response. One means to post-transcriptionally control cytokine expression is by modulating the stability of their mRNA via adenosine/uridine (AU)-rich elements (ARE), which are found within the 3’untranslated region (UTR) of several cytokine mRNAs. Although several ARE-interacting proteins (ARE-BPs) have been described, it is not clear how they regulate individual AREs of distinct cytokine mRNAs during the immune response. Methods Here we propose to use affinity purification coupled to mass spectrometry to analyze the dynamic interactions of selected cytokine ARE elements with ARE-BPs during an immune response. We chose to study the human interferon-beta (IFN-b) mRNA, which contains two instability determining sequences, an ARE in the 3’UTR and a coding region instability element (CIRD) within the open reading frame. We used 3’biotinylated IFN-b ARE RNA oligomers as affinity reagent to isolate ARE-BPs from total cell extracts of mock-treated or polyinosinic:polycytidylic acid (p(I:C)) stimulated cells. Isolated interacting proteins were analyzed via 1D shot gun mass spectrometry analysis. Results We could identify the human antigen R ( HuR ) as the most prominent IFN-b ARE binding protein. Previous studies have pointed to a role for HuR mRNA binding in antagonizing the rapid decay of some ARE-containing mRNAs, which correlates with reports of IFN-b message stabilization during the immune response. Conclusion The stability of cytokine mRNAs is highly regulated, as deregulation of cytokine production can have detrimental effects. Our work will shed new light on the dynamic interactions of cytokine ARE elements and their ARE-BPs during an immune response.