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Translational Regulation of Proto-oncogene MYC mRNA by AUF1: a Structure-Function Analysis

by
Estelle M. Ruidiaz-Santiago
B.S. University of Puerto Rico
Rio Piedras - 2003



Thesis Advisor: Gary Brewer, Ph.D.
Graduate Program in Molecular Genetics,
Microbiology & Immunology

Molecular Genetics, Microbiology & Immunology Department Conference Room 747, RWJMS Research Tower
Piscataway

Thursday, August 19, 2010
10:00 a.m.


Abstract

Sustained synthesis of MYC oncoprotein favors cell growth rather than differentiation, a hallmark of the neoplastic phenotype. An AU-rich element (ARE) within the 3’UTR of MYC mRNA controls its translation and posttranscriptional gene silencing. AUF1 is an ARE-binding protein family consisting of four isoforms (p37AUF1, p40AUF1, p42AUF1 and p45AUF1) that promote ARE-mediated translation of MYC mRNA. All isoforms contain two non-identical RNA-recognition motifs (RRMs) that are essential for ARE binding. As p40AUF1 is the predominant cytoplasmic isoform subject to regulatory changes in its phosphorylation, we performed structure-function analyses of p40AUF1 to examine its specific contribution to translation of MYC mRNA. Since domain-specific characteristics of p40AUF1 are well known, we created deletion and truncation mutants of p40AUF1, including evolutionarily conserved sequences. Assays of MYC ARE-binding affinities of these mutants revealed that regions outside the RRMs also contribute to high affinity ARE binding. Further characterization of p40AUF1 mutants included expression analyses of mutants in human tumor cell lines. Finally, we performed in vitro translation studies with rabbit reticulocyte lysates to recapitulate MYC translational control by p40AUF1. Our data suggest that exogenous p40AUF1 can stimulate in vitro translation of a MYC-ARE reporter RNA in this system. However, the effects we observed were somewhat variable with different preparations of recombinant p40AUF1 and reticulocyte lysate. Thus, while there are significant indications that p40AUF1 can activate translation of MYC mRNA in vivo and in vitro, further experiments will be required to resolve the variability issues related to preparation of p40AUF1 protein and reticulocyte lysate. Nonetheless, this system should ultimately prove valuable for dissecting the mechanisms by which AUF1 controls synthesis of the MYC oncoprotein to affect cellular proliferation versus differentiation decisions.


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