Determining the role for lincRNA-Cox2 in host defense against infection

Carpenter, Susan

Abstract

We have discovered that lincRNA-Cox2 can act as a critical regulator of innate immune genes. The exact mechanisms of action of this lincRNA remain unclear. Knockdown of lincRNA-Cox2 in vitro results in a decreased expression of pro-inflammatory genes while basal levels of interferon-stimulated genes (ISGs) are elevated.
In Aim1 we will determine the exact mechanisms utilized by lincRNA-Cox2 to control innate immune genes. We will use biochemical in vitro pull down assays to identify the critical regions or domains within lincRNA-Cox2 that control pro-inflammatory genes or ISG levels. We will perform RNA affinity purification to identify additional factors binding lincRNA-Cox2 to mediate its regulatory functions. In order to determine if lincRNA-Cox2 is critical for controlling host defense in vivo we have generated three murine models to test. We have a lincRNA-Cox2 knockout mouse in which the lincRNA locus has been replaced with a lacZ cassette. We have generated a splicing mutant mouse in which we removed the intron of lincRNA-Cox2 using Cas9 technology and finally we used the TARGATT system to generate a lincRNA- Cox2 over-expressing mouse. Preliminary data from our knockout mice suggests that lincRNA-Cox2 can mediate its effects on innate immune genes both in cis and in trans. We will use our additional models to answer critical questions about the functions of splicing and locus specific transcription and how they can impact lincRNA-Cox2's functions. We will perform LPS and E.Coli in vivo challenge models in order to determine if lincRNA-Cox2 is critical for controlling host defense in an entire organism. This project will allow us to address the importance of lincRNA-Cox2 in host defense and set in place a new layer of complexity and control in the RNA-immune-regulation of inflammation.

Public Health Relevance

The goal of this proposal is to further our understanding of how noncoding RNAs regulate gene expression in the context of infection. Specifically, we aim to undercover the mechanisms used by lincRNA-Cox2 to control inflammatory gene expression for host defense. !