Translational Silencing in Monocytes: Role of L13a

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DESCRIPTION (provided by applicant): Our long-term goal is to gain significant insight about the endogenous cellular strategy of targeted silencing of pro-inflammatory gene products. This proposal seeks to elucidate the mechanisms of translational silencing that may govern the natural resolution of the host response to inflammation. We have shown that interferon gamma (IFN-y), an inflammatory cytokine and a contributor to lesion progression in murine model of atherosclerosis, induces ceruloplasmin (Cp) synthesis in monocytes. Cp is an acute phase inflammatory protein that can oxidize LDL and may have an important role in monocytic cell mediated oxidative process in the vessel wall. In addition epidemiological studies also described Cp as an independent risk factor for cardiovascular disease. The synthesis of Cp protein in monocytes is terminated after about 16 hours of IFN-y treatment even in the presence of abundant Cp mRNA. We have described a novel translational silencing mechanism for this inhibition of Cp expression that depends on end-to-end circularization of the Cp transcript via interaction of the T-terminus with the 5'-translation-initiation complex. Cytosolic extracts made from monocytes after 16 hours of IFN-y treatment contain a protein complex(s), denoted IFN-Gamma Activated Inhibitor of Translation (GAIT) that binds to the 29-nt GAIT element present in the 3' untranslated region of Cp mRNA and silences its translation. In preliminary studies of this proposal, we have identified a protein, ribosomal protein L13a that specifically binds to the GAIT element and blocks in vitro translation of Cp. In addition, our work shows that prolonged treatment of monocytes with IFN-y causes release of L13a from the 60S ribosomal subunit and its phosphorylation that is required for silencing activity. The goal of this proposed study is to gain further insight in to the silencing mechanism by addressing the following Specific Aims: (1) How L13a inhibits translation initiation of Cp mRNA. We will use the reconstituted translational silencing system in reticulocyte lysate with recombinant L13a and centrifugal resolution of the different ribosomal subunit, to address this aim. (2) Determination of phosphorylation site and functional analysis of different domains of L13a. We will use phosphoamino acid analysis, mass spectrometry, site-directed and deletion-mutational analysis, in vivo and in vitro re-constitution of recombinant L13a phosphorylation, to address this aim. (3) Identification of other target mRNAs for L13a. We will address this aim by isolating mRNAs from the polysome obtained from the IFN-y activated monocytes as well as cells expressing siRNAs of L13a. The polysomal mRNAs will be subjected to RT-PCR and cDNA cloning and microarray analysis. Since inflammation is an important and obligatory component of initiation and progression of many diseases including atherosclerosis, we believe results obtained from the proposed study will yield significant insight into the translational silencing of pro-inflammatory gene products and may help to develop novel therapeutic agents that could effectively retard inflammation.