This proposal is directed to an important but very poorly understood mechanism of gene control, the targeted degradation of short-lived cytokine and proto-oncogene mRNAs in mammalian cells, which is promoted by an A+U-rich element (ARE) in the 3' untranslated region (3'UTR) of these mRNAs. This renewal application builds on our work during the previous project period and seeks to characterize the function of one ARE-binding protein known as AUF1, which promotes rapid decay of ARE-mRNAs.
Aim 1 will identify and characterize new AUF1 interacting proteins by mass spectrometry, verify interactions in vivo, and investigate the functional importance of these interactions in ARE-mRNA decay. Only several of the AUFl-interacting proteins have been identified to date. Since AUF1 lacks enzymatic activity, it is through interactions with other proteins that AUF1 facilitates or regulates decay of cytokine and proto-oncogene ARE-mRNAs.
Aim 2 will analyze the function of AUF1 in promoting or regulating the rapid turnover of endogenous and reporter ARE-mRNAs in isolated primary mouse spleen and thymus lymphocytic cells, where AUF1 is predominantly expressed. Our recent development of an AUF1 knockout mouse, and studies which localized AUF1 expression largely to splenic and thymic lymphocytes, makes it possible to investigate the molecular and biochemical functions of AUF1 in its authentic expressing cell type and organ.
Aim 3 will characterize the function of AUF1 in the mouse. There is little if any understanding of the role of AUF1 in animals. By comparing wild type and AUF1 knockout mice, studies will investigate the function of AUF1 in promoting or regulating the stability of lymphocytic cytokine mRNAs in the thymus and spleen. Studies will also examine the phenotype of the AUF1 knockout mouse in different relevant experimental settings.
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