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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
7R29CA054521-04
Application #
3460241
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1991-04-15
Project End
1996-03-31
Budget Start
1993-09-20
Budget End
1994-03-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Yang, H; Duckett, C S; Lindsten, T (1995) iPABP, an inducible poly(A)-binding protein detected in activated human T cells. Mol Cell Biol 15:6770-6
Katz, D A; Theodorakis, N G; Cleveland, D W et al. (1994) AU-A, an RNA-binding activity distinct from hnRNP A1, is selective for AUUUA repeats and shuttles between the nucleus and the cytoplasm. Nucleic Acids Res 22:238-46
Lindsten, T; Lee, K P; Harris, E S et al. (1993) Characterization of CTLA-4 structure and expression on human T cells. J Immunol 151:3489-99
Mao, X; Green, J M; Safer, B et al. (1992) Regulation of translation initiation factor gene expression during human T cell activation. J Biol Chem 267:20444-50
Bohjanen, P R; Petryniak, B; June, C H et al. (1992) AU RNA-binding factors differ in their binding specificities and affinities. J Biol Chem 267:6302-9