Research Interests
Post-transcriptional Gene Regulation
Post-transcriptional processes play a crucial role in controlling gene expression in all organisms. Our research is aimed at elucidating post-transcriptional gene regulation in eukaryotic and bacterial cells. We are particularly interested in the proteins, RNA elements, and molecular mechanisms that govern mRNA degradation, a key regulatory process that directly impacts gene expression through its influence on mRNA abundance. We are also conducting studies aimed at elucidating how microRNAs regulate gene expression in mammalian cells via changes in mRNA translation and stability. Finally, we have developed a set of broadly applicable genetic methods for rapidly cloning and characterizing proteins important for post-transcriptional gene regulation, and we are using these and other methods to investigate how such proteins (e.g., HIV-1 Rev) assemble on their RNA targets and control gene expression.
Figure 1. 5' untranslated region of E. coli rne mRNA.
mRNA degradation
In bacteria, the cytoplasmic lifetimes of mRNAs can differ by more than an order of magnitude, with profound consequences for gene expression. Despite the regulatory significance of mRNA decay, much remains unknown about the molecular mechanisms that govern this process. We have shown that elements in the 5' untranslated region can determine the cytoplasmic lifetime of an entire mRNA molecule by controlling the accessibility of downstream sites to ribonuclease cleavage. We are now using a variety of biochemical and molecular biological approaches to investigate the mechanism(s) underlying this 5'-end dependence. Much of our research in this area has focused on RNase E, a key endonuclease whose action is thought to trigger the degradation of most mRNAs in E. coli.
Figure 2. Regulation by microRNAs.
microRNA function
The cells of most eukaryotic organisms contain many different microRNAs, short RNA molecules that function in gene regulation. In animal cells, microRNAs repress gene expression by annealing to mRNAs to which they are imperfectly complementary. Our studies have indicated a role for the microRNA miR-125b in mammalian neuronal development, as its increased concentration when mouse embryonal carcinoma cells are induced to differentiate into neurons contributes to the downregulation of several genes. Repression by miR-125b and other microRNAs is a consequence of reductions in both translation efficiency and mRNA stability. Our current research is aimed at understanding the molecular mechanisms by which microRNAs downregulate gene expression via these two mechanisms.
Figure 3. Structural model of HIV-1 Rev bound to RRE stem-loop IIB.
RNA-protein recognition
Many of the proteins that control gene expression post-transcriptionally are RNA-binding proteins. Notwithstanding their biological and medical importance, much remains to be learned about the mechanisms by which these proteins recognize and assemble on their RNA targets. We are currently examining two such polypeptides, HIV-1 Rev and Drosophila Pumilio, in an effort to better understand how these important regulatory proteins interact with RNA. Most of our studies of Rev have focused on its structure and the architecture of its multimeric assembly on HIV RNA, which is crucial for the efficacy of Rev in mediating the post-transcriptional transition from early to late viral gene expression. In the case of Pumilio, we are investigating whether the modular manner in which this protein recognizes the sequence of its RNA ligand can be manipulated to alter its RNA-binding specificity.