RNA is a master regulator of gene expression, and disruption of RNA-dependent events can lead to devastating human diseases, such as viral infections and cancer. The main research goal of the laboratory is to better understand the role of RNA in the regulation of gene expression and help the development of RNA-based tools for biochemical and therapeutic applications. We investigate fundamental aspects of RNA structure, function and engineering using unique and innovative approaches that combine biochemical and biophysical methods, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and bioinformatics.
The research projects are grouped along 4 main axes: 1) Structure, function and engineering studies of ribozymes; 2) Structural investigations of riboswitches; 3) Structural and functional studies of protein / microRNA interactions in the regulation of gene expression; and 4) Structural and functional studies of interactions regulating the human RNA polymerase II (RNAPII) CTD phosphatase FCP1.
1) Structure, function and engineering of ribozymes. The discovery of ribozymes increased our appreciation of the functional importance of RNA in biology. Several naturally occurring ribozymes, including the hammerhead, hairpin and Neurospora VS ribozymes, catalyze phosphodiester bond cleavage in the absence of proteins. Some of these ribozymes have been engineered to recognize and cleave specific RNA sequences. Thus, these ribozymes represent powerful tools for gene inactivation, particularly for anticancer and antiviral therapies. In addition, activatable ribozymes can be engineered to function as ligand-controlled genetic switches or RNA-based biosensors for a wide range of potential applications, including medical diagnostics.
The VS ribozyme is currently the only known nucleolytic ribozyme for which there is no complete three-dimensional structure. Such a structure is needed in order to gain insights into its catalytic mechanism, better understand its unusual mode of substrate recognition and assist in engineering efforts.
We are currently pursuing a modular approach to structurally characterize the VS ribozyme, which consists of determining NMR structures of domains that are relevant to its function. In addition, we are using existing structural knowledge to engineer VS-derived ribozymes with novel functions.
2) Structural investigations of riboswitches. Riboswitches are functional RNA elements typically located in the 5'-untranslated region of mRNA and control gene expression in the absence of proteins. Riboswitches are composed of two domains: an aptamer domain and an expression platform. The aptamer domain binds to a metabolite ligand and the expression platform controls gene expression by adopting two mutually exclusive conformations depending on ligand binding. In one conformation, the gene is expressed and in the other it is repressed, providing an on/off RNA switch in gene regulation.
Over 20 families of riboswitches have been identified to date, which recognize a diverse set of metabolites, including nucleotides, amino acids, sugars, vitamin cofactors, and metal ions. Although riboswitches have been identified in a number of organisms, including fungi and plants, they are very important in bacteria where they account for about 2–3% of genetic. Given their importance in controlling bacterial homeostasis, metabolite-sensing riboswitches are being considered as antimicrobial targets, and agonist ligands have been employed to knockdown the expression of associated genes.
We are carrying structural studies of riboswitches, such as the purine riboswitches, to provide atomic details of the ligand binding sites, and such information will be useful for the design of novel antibiotics against pathogenic bacteria.
3) Structure and function of protein / microRNA interactions. MicroRNAs are small non-coding RNAs [20-30 nucleotides (nt)] that constitute one of the largest families of transacting gene regulatory molecules in multi-cellular organisms. In RNA interference, the miRNAs base pair with complementary messenger RNAs (mRNAs) and thereby inhibit translation. It is known that mammalian miRNAs largely target mRNAs of developmental genes and that their misregulation is linked to a variety of human diseases, including several forms of cancer.
Among those miRNAs that have been associated with cellular differentiation and cancer, the let‑7 family of miRNAs has been the most thoroughly investigated. Previous studies have demonstrated that the Lin28 protein regulates let-7-g biogenesis via a direct interaction with the precursor forms of the let‑7g miRNA (pri-let-7-g and pre‑let‑7g).
Although various studies point to the importance of the pre‑let‑7 loop for Lin28 recognition, the detailed molecular mechanisms through which Lin28 and pre-let-7g interact are currently unknown. We are currently examining the specific interactions involving the Lin28 protein and the pre-let-7g miRNA using biochemical, biophysical and structural methods.
4) Structure and function of interactions regulating FCP1. In eukaryotes, the phosphorylation state of the carboxyl-terminal domain (CTD) of the largest subunit of RNAPII plays a critical role in transcription regulation. FCP1 is a highly conserved and essential CTD phosphatase that associates with RNAPII and regulates its activity. It is clearly established that numerous proteins interact with FCP1 to regulate its functions as a phosphatase and as a positive elongation factor.
Our previous NMR studies have provided a detailed view of the interactions involving the central and C-terminal domains of FCP1 and the RAP74 subunit of the general transcription factor TFIIF. Using biochemical methods, we have characterized the interactions of the central domain of FCP1 with the HIV-1 Tat protein. We have also analyzed the effect of the phosphorylation of FCP1 by casein kinase 2 (CK2) on several interactions.
We continue to combine biochemical and structural methods to gain a better understanding of the macromolecular interactions that regulate FCP1 functions. More specifically, we are pursuing: 1)
biochemical and structural characterization of the role of CK2 phosphorylation in binding of FCP1 to target proteins; 2)
biochemical and structural interactions of FCP1 with nucleic acid; and 3)
identification and characterization of novel FCP1-associated molecules.