Scott A. Strobel
RNA Structure and Cellular Localization
Overview: The research efforts of my lab focus on the biochemical basis of RNA structure and function. This work includes three major projects, (i) characterization of the 5-methyl cytidine DNA demethylase postulated to include an RNA component, (ii) determination of the RNA tertiary structure that defines a molecular zip code for cytoplasmic RNA localization during oogenesis, and (iii) developing nucleoside analogs to define the specific chemical funtional groups within an RNA that are important for its function. These projects span a wide breadth of techniques ranging from microinjection to chemical organic synthesis. The central theme of the work is to determine the relationship between the structure of the RNA and its cellular function, whether that function be chemical catalysis or cellular localization.
1. Characterization of the 5-methyl Cytidine Demethylase--A Putative Ribozyme. DNA demethylation is a cellular phenomenon that is poorly understood, despite the fact that it is fundamental to a broad diversity of human disease mechanisms. Methylation at the 5-position of cytidine within the CpG dinucleotide plays a critical role in the repression of gene expression in animal cells. DNA methylation is maintained by a balance between methylation and demethylation of each site in the genome. The pattern of methylation is cell type and developmental stage specific. While the methyltransferase has been well characterized, very little is understood about the demethylation activity. Recently Howard Cedar's lab at Hebrew University in Israel has shown that the demethylation activity present in extracts from a wide variety of cell types might be mediated by an RNA. The goal of our work is to clone, characterize, and determine the biological relevance of the 5-methyl cytosine demethylase from murine fibroblasts.
2. An RNA Molecular Zip Code for Cellular Localization. The object of this research project is to define the molecular basis of mRNA localization, an important mechanism for regulating gene expression in asymmetric germ and somatic cells. We hope to demonstrate that the localization signal encoded in the 3'-untranslated region of the mRNA is defined by an RNA tertiary structure. The work focuses on two cytoplasmically localized mRNAs in Xenopus oocytes, Vg1 and Xcat-2. Through the use of in vivo combinatorial selection we are trying to generate a family of related, but unequal, sequences that are capable of directing the mRNA to the proper cellular location. By identifying the conserved sequence elements within the library of related RNAs, we will be able to identify the secondary and tertiary structural elements important for cellular localization. This information will be complemented by chemical modification data including the extensive use of photocrosslinking agents to provide three dimensional constraints for modeling the RNA structure. This work is being performed in Xenopus oocytes because they are amenable to biochemical manipulation, however, the information gained about RNA localization is likely to be applicable to many asymmetric cell types that localize their RNAs.
3. Defining the Chemical Basis of RNA Structure and Catalysis. Formation of higher order RNA structure is achieved by long range tertiary interactions. These interactions are mediated by specific functional groups within the RNA. We are synthesizing a series of nucleoside analogs that will allow us to rapidly identify the chemical groups that are important for RNA structure and function. We have synthesized the 5'-O-(1-thio-triphosphates of several nucleoside derivatives. These phosphorothioates are oxidatively labile and allow us to map the sites where incorporation of the modified nucleoside is detimental to RNA function. Using these reagents we can rapidly identify the 2'-hydroxyls, exocyclic amines, carbonyls, and imino groups that are important to activity within an RNA. Our initial focus is on the Tetrahymena group I intron. The results will provide excellent support for the phylogenetic information available for the group I introns. A portion of the data can be interpreted at high resolution using the crystal structure available for a subdomain of the ribozyme. We are also beginning work in other systems. We have established collaborations to map the splice leader RNA, U6 snRNA, RNaseP and the decoding site in rRNA. We will also use these reagents to characterize the systems described in the first two projects after they have been more completely developed.