Gene expression can be regulated at the mRNA level through alterations in transport, translational efficiency and stability. The rate of mRNA decay, in addition to the rate of transcription, determines cytoplasmic abundance of mRNAs. Regulation of mRNA decay rates is therefore an important control point in gene expression. It is well established that different mRNAs display diverse half-lives in eukaryotic cells, ranging from minutes for many inflammatory cytokine and growth factor mRNAs, to many hours for most other mRNAs. Acute pro-inflammatory cytokine responses are induced in tissues by ionizing radiation (IR) exposure, particularly in macrophages and other immune cells, and the pathological inflammatory response is a major complicating affect of IR during radiotherapy and accidental exposure. Despite the obvious importance for understanding how IR promotes a pathological inflammatory response, there has been remarkably little research conducted to characterize the mechanism by which this occurs. This application is directed to understanding the molecular mechanism by which IR promotes overexpression of pro-inflammatory cytokines, focusing on its ability to inhibit the normal rapid degradation of inflammatory cytokine mRNAs by antagonizing the activity of pro-decay factor, AUF1.
Aim 1 will use immortalized wild type and AUF1-/- macrophages and mass spectrometry to identify and characterize IR-inducible AUF1 interacting proteins that are likely involved in mediating IR control of the inflammatory response through regulation of AUF1 activity. To obtain information relevant to the clinical setting, additional studies will examine the affect of multiple lower dose fractions of IR on AUF1-mediated decay of inflammatory ARE-mRNAs and AUF1-interacting proteins. Studies will then identify proteins whose binding to AUF1 is lost or gained with single high dose and multiple fractionated lower dose IR- treatment. The in vivo interaction of these proteins with AUF1 will then be verified under physiologically relevant conditions.
Aim 2 will characterize the function of IR-mediated AUF1 binding protein interactions on the rapid decay of endogenous pro-inflammatory cytokine ARE-mRNAs. Studies will use immortalized wild type and AUF1-/- macrophages, as well RNA silencing and cDNA overexpression of interacting proteins, to fully characterize their molecular functions in IR-mediated stabilization and overexpression of inflammatory cytokine mRNAs.
Aim 3 will identify the major endogenous inflammatory cytokine ARE-mRNAs that are regulated by IR through control of AUF1 activity. Studies will identify global ARE-mRNA targets of AUF1 activity in untreated and IR treated immortalized wild type and AUF1-/- macrophages, and determine the extent to which other ARE-binding proteins such as TTP, KSRP and stabilizing protein HuR are involved in IR-mediated stabilization of inflammatory cytokine ARE-mRNAs.
Aim 4 will characterize the molecular mechanism by which IR inhibits the decay of inflammatory ARE- mRNAs by acting on AUF1 and the key AUF1 interacting proteins identified. Studies will also determine whether IR blocks AUF1 function by acting on P-body formation and function, and/or on the exosome, a ribonuclease organelle-like structure that is involved in the degradation of short-lived mRNAs.
The purpose of this grant application is to understand the mechanism by which inflammatory cytokines are controlled by the rapid degradation of their encoding mRNAs and how this is regulated by ionizing radiation. Several proteins have been shown to bind specifically to these mRNAs and to promote their degradation. This application seeks to define the mechanism by which one of those proteins known as AUF1, controls the rapid degradation of the key inflammatory cytokines in response to ionizing radiation.
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