Hematopoietic cells are produced and destroyed continuously under precise control. Growth and differentiation of these cells requires exact modulation by growth factors. Major site of regulation of production of cytokines is through stabilization of their transcripts. Tumor necrosis factor (TNF), interleukin-lbeta (IL-1beta), and phorbol diester (TPA) cause accumulation of GM-CSF in cells through stabilization of its message. We will use GM-CSF RNA produced in fibroblasts (W138 cells) as a paradigm for our studies of regulation of stability of transcripts of cytokines. The motif AUUUA is repeated a number of times in the 3' untranslated region (UTR) of RNAs encoding short-lived cytokines and oncogenes; and they cause RNA destabilization when attached to a reporter gene such as rabbit beta-globin (RbetaG).
Specific Aim 1 will determine the effect of different numbers and locations of AUUUA sequences on stability of RbetaG reporter gene transcripts in cells with or without stimulation by either TNF, IL-1beta or TPA. In addition, studies have suggested that AUUUA sequences may impair efficient translation of transcripts containing these sequences.
Specific Aim 2 will define the importance of number and location of AUUUA sequences as well as various regions of 3'UTR of GM-CSF RNA in translational control using our RbetaG reporter genes.
Specific Aim 3 will develop and use an in vitro technique to assay ani study the functional significance of ribonuclease and protecting factors that interact with regions of GM-CSF mRNA and that may be involved in modulation of GM-CSF RNA stability.
Specific Aim 4 will use gel retardation to identify and investigate the trans-elements (ribonuclease and protecting factors) and cis-elements (specific regions of GM-CSF RNA), involved in modulation of stability of GM-CSF RNA.
Specific Aim 5 will purify, clone and characterize GM-CSF RNA binding proteins. Characterization of these proteins will use specific antibodies to study protein levels, and will use expression and antisense vectors to study function of the RNA binding protein. These studies will provide a detailed understanding of a major regulatory pathway of GM-CSF production. In addition, the knowledge that is gained in these experiments should be applicable to regulation of many transiently expressed cytokines and oncogenes. This knowledge may allow us to understand more clearly how to control production of hematopoietic cells.
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