UC Berkeley

Abstract

The Regulation of TLR7 and TLR9 in the Prevention of Autoimmunity

By

Zachary Robert Newman

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Gregory M Barton, Chair

Recognition of nucleic acids is an evolutionarily conserved trait of the immune system that enables detection of a diverse array of microbial species with relatively few receptors. However, the inappropriate recognition of self-derived nucleic acids is well documented for its role in autoimmune diseases. Two such receptors that recognize nucleic acids, Toll-like receptor (TLR) 7 and TLR9, are highly regulated in order to maintain the proper discrimination between self and non-self. In this dissertation I discuss the background of what is known about TLRs, with an emphasis on nucleic acid sensing TLRs and evidence for their involvement in autoimmune disease. I also provided an introduction to the phenomenon of codon usage bias and discuss the debate surrounding its functional significance. I then present our findings that codon bias may be playing a significant role in the balance of protein levels between TLR7 and TLR9. In an endogenous context, we observe a markedly different measured translational efficiency for TLR7 compared to TLR9, correlating with their degree of codon bias. Codon optimization of TLR7’s coding sequence dramatically increases the translation efficiency and subsequent protein levels in an exogenous setting. Furthermore, by scaling the level of optimization we reveal how the different mechanisms of transcription, mRNA stability, and translation, all of which contribute to overall protein levels, are affected by codon bias. In contrast to the exogenous setting, codon optimization of TLR7 at the endogenous locus did not result in an increase in TLR7 function suggesting additional levels of regulation may be involved. In this dissertation I also introduce a novel method to study the functional significance of misregulated localization of nucleic acid sensing TLRs in an endogenous context. Our studies introduced a conditional mutant allele of TLR9 that is capable of signaling from the cell surface. The mutant is well expressed in a controlled, cell-specific manner with preliminary results suggesting potential autoimmune consequences. Combined, our studies significantly add to the literature by illuminating the importance of two distinct mechanisms regulating nucleic acid sensing TLRs in the prevention of autoimmunity.