The objective of this project is to identify and characterize lymphokine-specific RNA-binding proteins that may serve to regulate post-transcriptional regulation of lymphokine gene expression in the cytoplasm.
The specific aims are as follows: 1) to define structural motifs, involved in binding of lymphokine mRNAs to cytoplasmic proteins. For instance, all lymphokines and other unstable mRNAs share a common motif, AUUUA, in their 3' untranslated region. Preliminary data show that extracts from normal human T cells contain two separate AUUUA-binding activities. One binds to AU-rich regions in all unstable mRNAs. The other specifically binds only those present in lymphokine mRNAs. By RNase protection assays and mutagenization of the sequence involved the exact sequence involved in these two binding complexes will be determined. Additional studies will be performed to determine if other lymphokine RNA sequence motifs bind to cytoplasmic proteins; 2) to identify trans-acting factors that interact with conserved RNA sequence motifs: First the molecular components of the AUUUA-binding complexes described above will be characterized. If the binding complexes identified consist of single proteins, these proteins will be cloned using expression libraries; 3) development of an in vitro RNA degradation assay: Previously described methods will be employed to purify polysomes from human T cells to be used in in vitro decay assays. It will be tested whether the purified RNA-binding complexes mentioned above will affect polysomal RNA degradation either specifically or nonspecifically; 5) to characterize RNase involved in rapid degradation of lymphokine mRNAs: The mechanism of endogenous lymphokine mRNA degradation will be analyzed using previously described assays. To define the cellular RNase involved in the degradation process, the effects of lymphokine-specific RNA binding proteins on RNase activities required for lymphokine degradation will also be examined. Through these specific aims, characteristics of lymphokine-specific mRNA binding proteins and their potential role in determining mRNA stability will be established.
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