RBPs are pivotal regulators of mRNA metabolism, influencing all aspects of post-transcriptional gene control: pre-mRNA splicing, mRNA transport, mRNA stability, and translation. We study if a given RBP associates with a given mRNA by a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules (ribonucleoprotein immunoprecipitation). To investigate RBP function in mammalian cultured cells, we employ approaches such as RBP silencing, RBP overexpression, and the identification of RBP-associated mRNAs using microarrays. We investigate whether RBPs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs.? ? During this funding period, we have completed two main projects. In one project, we systematically analyzed the interactions of six major RBPs (HuR, AUF1, NF90, KSRP, TIAR, and TIA-1) with the mRNAs that encode these RBPs. We reported that each RBP associates with its own mRNA and uncovered an extensive network of cross-regulation among these RBPs and the mRNAs that encode them. In the other main project, we reported that HuR is phosphorylated by the kinase Cdk1 (Cdc2); Cdk1 phosphorylates HuR at S202 (a residue located within a region important for controlling the subcellular levels of HuR) and together with 14-3-3 proteins, it helped to retain HuR in the nucleus.? ? Ongoing work is examining the subcellular localization of HuR by post-translational modification at S242 (also located within the region that modulates HuR subcellular distribution), and the influence of ubiquitination upon the overall HuR levels in cells responding to heat shock. Other studies are seeking to identify the subsets of mRNAs that are regulated by RBPs NF90 and AUF1.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000392-01
Application #
7732228
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2008
Total Cost
$592,080
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Abdelmohsen, Kotb; Kim, Mihee M; Srikantan, Subramanya et al. (2010) miR-519 suppresses tumor growth by reducing HuR levels. Cell Cycle 9:1354-9
Figueroa, Angelica; Kotani, Hirokazu; Toda, Yoshinobu et al. (2009) Novel roles of hakai in cell proliferation and oncogenesis. Mol Biol Cell 20:3533-42
Mazan-Mamczarz, Krystyna; Kuwano, Yuki; Zhan, Ming et al. (2009) Identification of a signature motif in target mRNAs of RNA-binding protein AUF1. Nucleic Acids Res 37:204-14
Brennan, Sarah E; Kuwano, Yuki; Alkharouf, Nadim et al. (2009) The mRNA-destabilizing protein tristetraprolin is suppressed in many cancers, altering tumorigenic phenotypes and patient prognosis. Cancer Res 69:5168-76
Masuda, Kiyoshi; Abdelmohsen, Kotb; Gorospe, Myriam (2009) RNA-binding proteins implicated in the hypoxic response. J Cell Mol Med 13:2759-69
Costantino, Christina L; Witkiewicz, Agnieszka K; Kuwano, Yuki et al. (2009) The role of HuR in gemcitabine efficacy in pancreatic cancer: HuR Up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase. Cancer Res 69:4567-72
Ale-Agha, Niloofar; Galban, Stefanie; Sobieroy, Christiane et al. (2009) HuR regulates gap junctional intercellular communication by controlling beta-catenin levels and adherens junction integrity. Hepatology 50:1567-76
Gorospe, Myriam (2009) Ribonucleoprotein dynamics connects mRNA networks with drug mechanisms. Mol Syst Biol 5:289
Ishmael, Faoud T; Fang, Xi; Galdiero, Maria Rosaria et al. (2008) Role of the RNA-binding protein tristetraprolin in glucocorticoid-mediated gene regulation. J Immunol 180:8342-53
van der Brug, Marcel P; Blackinton, Jeff; Chandran, Jayanth et al. (2008) RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways. Proc Natl Acad Sci U S A 105:10244-9

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