RNA-Binding Proteins that Modulate Proliferative and Stress-Response Genes

National Institute On Aging

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Abstract

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.