Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3′UTR

Abstract

Innate immune signaling is the front line of defense against pathogens, leading to an appropriate response of immune cells upon activation of their pattern recognition receptors (PRRs) by microbial products, such as Toll-like receptors (TLRs). Apart from transcriptional control, gene expression in the innate immune system is also highly regulated at the post-transcriptional level. miRNA or RNA-binding protein can bind to the 3′ untranslated region (UTR) of target mRNAs and affect their mRNA stability and translation efficiency, which ultimately affects the amount of protein that is produced. In recent years, a new group of PRRs, the Nod-like receptors (NLR) have been discovered. They often cooperate with TLR signaling to induce potent inflammatory responses. Many NLRs can form inflammasomes, which facilitate the production of the potent pro-inflammatory cytokine IL-1β and other inflammatory mediators. In contrast to TLRs, the importance of post-transcriptional regulators in the context of inflammasomes has not been well defined. This chapter describes a series of experimental approaches to determine the effect of post-transcriptional regulation for a gene of interest using the best-studied NLR, NLRP3, as an example. To start investigating post-transcriptional regulation, 3′UTR luciferase experiments can be performed to test if regulatory sequences in the 3′UTR are functional. An RNA pull-down approach followed by mass spectrometry provides an unbiased assay to identify RNA-binding proteins that target the 3′UTR. Candidate binding proteins can then be further validated by RNA immunoprecipitation (RNA-IP), where the candidate protein is isolated using a specific antibody and bound mRNAs are analyzed by qPCR.