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Gary Brewer, PhD

Office: 732-235-3473
Fax: 732-235-5223

Office: RWJMS 736
Lab: RWJMS 733



Donnini, M., Lapucci, A., Papucci, L., Witort, E., Jacquier, A., Brewer, G., Nicolin, A., and Capaccioli, S. Identification of Tino: A new evolutionarily conserved BCL-2 AU-rich element RNA-binding protein. J Biol Chem, 2004; 279:20154-20166.

Liao, B., Patel, M., Hu, Y., Charles, S., Herrick, D.J., and Brewer, G. Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an IGF-II-dependent pathway in human K562 leukemia cells. J Biol Chem, 2004; 279:48716-48724.

Liao, B., Hu, Y., Herrick, D.J., and Brewer, G. The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during proliferation of human K562 leukemia cells. J Biol Chem, 2005; 280:18517-18524.

Sommer, S., Cui, Y., Brewer, G., and S.A.W. Fuqua. The c-Yes 3’-UTR contains adenine/uridine-rich elements that bind AUF1 and HuR involved in mRNA decay in breast cancer cells. Steroid Biochem Mol Biol, 2005; 97:219-229.

Knapinska, A.M., Irizarry-Barreto, P., Adusumalli, S., Androulakis, I., and Brewer, G. Molecular mechanisms regulating mRNA stability: Physiological and pathological significance. Curr Genomics, 2005; 6:471-486.

Lal, A., Abdelmohsen, K., Pullman, R., Kawai, T., Yang, X., Brewer, G., and Gorospe, M. Posttranscriptional derepression of GADD45a by genotoxic stress. Mol. Cell, 2006; 22:117-128.

Chesoni, S. and Brewer, G. 2006. A003244 HNRPD Molecule Page. AfCS-Nature

Gupta, M. and Brewer, G. MicroRNAs: New players in an old game. Proc Natl Acad Sci USA, 2006; 103:3951-3952.

Lab Staff

Jennifer Defren

Graduate Student

Frances Gratacos

Graduate Student

Malavika Gupta

Graduate Student

Patricia Irizarry

Graduate Student

Anna Knapinska

Graduate Student

Estelle Ruidiaz

Graduate Student

Riza Ysla

Graduate Student

Research Interests

The control of mRNA turnover is an important means of regulating both the level and timing of gene expression. Messenger RNAs like c-myc, c-fos and GM-CSF whose protein products influence proliferation and differentiation, are relatively unstable, with half-lives of an hour or less. As a result of their instability, modest changes in their turnover rates affect their steady-state levels over a relatively short time period. The short-term regulation ensures that the concentrations of these mRNAs are maintained within a limited range. The necessity of this precise regulation is consistent with their inappropriate expression interfering with proliferation and differentiation.

Rapid mRNA decay results from cis-acting elements present in labile mRNAs. For example, many mRNAs encoding oncoproteins, cytokines, and G protein-coupled receptors are labile due to the presence of A+U-rich elements (AREs) in their 3′ -UTRs. We are interested in why some mRNAs are very labile, what trans-acting factors effect rapid mRNA decay, and how these factors are regulated. For these reasons we developed and have utilized a cell-free mRNA decay system to biochemically dissect the turnover machinery. Using this system we identified, purified and molecularly cloned an RNA-binding protein, AUF1, which affects ARE-directed mRNA decay via its high-affinity binding to AREs. cDNA and genomic cloning experiments indicate that alternative pre-mRNA splicing generates four AUF1 isoforms with apparent molecular weights of 37, 40, 42 and 45 kD. Antisera to AUF1 demonstrate that they are phosphorylated, are located in the nucleus and cytoplasm, and form one or more complexes with at least five additional polypeptides. We have a number of projects ongoing in my lab, and I will describe these in detail below.

(1) My lab is currently utilizing the cell-free mRNA decay system to dissect how AUF1 -ARE interactions target the mRNAs for rapid decay. Recent results indicate that targeting of AUF1 to the proteasome is necessary for ARE-directed mRNA decay. Additionally, we are purifying the AUF1-associated proteins mentioned above and have identified a number of these: translation initiation factor eIF4G, the heat shock proteins hsp/hsc70, and poly(A)-binding protein. We plan to use purified components in the cell-free system to examine how they function in concert with AUF1 to effect ARE-directed mRNA turnover of c-myc and c-fos mRNAs. The results from this study have clear implications for elucidating how alterations in posttranscriptional control of gene expression may lead to tumorigenesis.

(2) The biosynthesis of each AUF1 isoform is controlled by alternative pre-mRNA splicing, which is regulated developmentally and perhaps also by tissue specificity. This has profound consequences for the stability of ARE-containing mRNAs. For example, we have found that activated mononuclear cells (MNC) from newborn children express the 37 and 40 kD isoforms to high levels, while those from adults express predominately the p42 and p45 isoforms. The half-life of GM-CSF mRNA in newborn MNC is at least 4-fold shorter than the half-life observed in adult MNC, suggesting that the 37 and 40 kD isoforms effect a more rapid turnover rate than p42 or p45. In support of this hypothesis, we have identified a cell line which expresses reduced levels of p37 and p40, and GM-CSF mRNA is quite stable in these cells. My lab is currently utilizing this cell line to examine the effects of ectopic expression of p37 and p40 on ARE-directed mRNA decay. Since myelopoiesis in the newborn is developmentally immature compared to the adult, the results of these studies should have ramifications for understanding misregulation of neonatal phagocytic immunity.

(3) AUF1 levels are elevated in hearts of patients with congestive heart failure compared to normal hearts. By contrast, the levels of ß1-adrenergic receptor (ß1AR) mRNA and protein are lower in failing hearts. Levels of ß2AR mRNA and protein are not different between normal and failing hearts, however. Since we have found that AUF1 binds the 3′ -UTR of ß1AR mRNA with an affinity 25-fold greater than that for ß2AR mRNA, our hypothesis is that elevated levels of AUF1 selectively increase the turnover rate of ß1AR mRNA, which in turn results in lower levels of ß1AR mRNA and protein. Because the ß1AR protein is essential for cardiac output, we believe that elevated levels of specific AUF1 isoforms in the failing heart is detrimental to cardiac function. My lab is currently developing a transgenic model of cardiac AUF1 overexpression to further address this relationship.

(4) A MAP kinase referred to as p38/RK (reactivating kinase) controls inflammatory cytokine biosynthesis, and we have some evidence that AUF1 may be a target of this signal transduction pathway. Proinflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNFα) are produced by monocytes in response to adherence or to inflammatory stimuli such as bacterial lipopolysaccharide (LPS). IL-1ß and TNFα biosyntheses are regulated at both the transcriptional and posttranscriptional levels. At the posttranscriptional level, adherence rapidly induces mRNA stabilization. Additionally, the posttranscriptional effects appear to require the AREs present in the IL-1ß and TNFα mRNAs. We found that AUF1 in extracts of nonadherent monocytes binds cytokine AREs; binding is abolished in extracts prepared from adherent cells. This correlates with adherence-induced stabilization of mRNAs. Moreover, AUF1 binding activity in monocyte extracts is sensitive to protein kinase inhibitors. My lab has recently found alterations in phosphorylation of AUF1 with adherence concomitant with mRNA stabilization. We have also found that phosphorylation alters the ARE-binding properties of AUF1. Currently, we are mapping phosphorylation sites so that we can examine the effects of phosphorylation site mutants on mRNA decay in cells.

(5) Finally, we are utilizing physical methods to dissect the molecular details of the binding of AUF1 to its cognate RNA sequences. In particular we are evaluating AUF1:ARE binding in solution by fluorescence anisotropy measurements. Our results so far indicate sequential, protein-dimer binding to RNA. We are now exploring the kinetics of these processes.