Translational Silencing in Monocytes: Role of L13a

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DESCRIPTION (provided by applicant): Atherosclerosis is a pathogenic consequence of uncontrolled inflammation of the resident cells of vessel wall. Using cell-based model we showed that ribosomal protein L13a-dependent translational silencing pathway could terminate the expression of a group of inflammatory genes. In this proposal we will test whether this mechanism can resolve inflammation. We have demonstrated that IFN-3 induced synthesis of Ceruloplasmin (Cp) and a group of other inflammatory proteins in monocytic cells is under translational control. Further, we discovered a crucial role of ribosomal protein L13a and its release from 60S ribosome in the formation of IFN- Gamma-Activated Inhibitor of Translation (GAIT) complex. The translational silencing mechanism relies on the recognition of L13a-dependent GAIT complex by the GAIT element present in the 3< untranslated region (UTR) of the target mRNAs. This is based on our observation that shows the depletion of L13a by RNA silencing can overcome the translation inhibition of these target mRNAs. To our surprise, the depletion of L13a had no effect on overall protein synthesis, however several Internal Ribosome Entry Site (IRES) dependent translation were inhibited. This proposal wants to test the hypothesis that L13a-dependent translational silencing of these target mRNAs coding a cluster of inflammatory proteins may serve as an endogenous defense mechanism against uncontrolled inflammation and atherosclerosis. On the other hand, the role of L13a may be confined to specialized rather than overall function of the ribosome. We will test our hypothesis by pursuing the following three specific aims: (1) Analysis of the L13a regulated and inflammation responsive post-transcriptional operon in IFN-3 activated monocytes. In these aim we will undertake a comprehensive analysis of the new target mRNAs the member of the post-transcriptional operon by assessing the de novo translation, identifying the cis-active elements and the ribonucleoprotein complex recognized by these elements of the new target mRNAs. (2) Studies on the physiological role of the GAIT complex mediated translational silencing. We have generated mice harboring the conditional null allele (floxed allele) of L13a. Crossing LysMCre mouse with the floxed mouse will generate the macrophage specific knockout of L13a. Macrophages isolated from these mice will be tested ex-vivo for translational silencing. On the other hand these mice will be challenged with the inducer of inflammation and the response will be examined on multiple levels. (3) Role of L13a in ribosome function and IRES activities. In this aim using a combined approach involving ribosome fractionation, immunoblot analysis and in vitro ribosome binding we will determine the ribosome-binding site of L13a. We will use the translation competent extract of L13a depleted cell to determine the mechanism of L13a dependence of the IRES activities. We believe these studies will discover novel cellular targets for new generation of anti-inflammatory small molecules against atherosclerosis and other inflammatory diseases. PUBLIC HEALTH RELEVANCE: Uncontrolled inflammation is the cause of many diseases such as atherosclerosis or cardiovascular disease. This research will uncover the insight about the endogenous cellular mechanisms to control the expression of inflammatory molecules and help to generate novel therapeutic agents against inflammatory diseases.