Structural and Chemical Biology of RNA Processing
Research in my laboratory is focused on the chemistry and biology of nucleic acids with an emphasis on biologically important reactions involving RNA. Large RNAs and complex ribonucleoprotein machines such as the spliceosome and ribosome play a key role in constitutive and regulated cellular processes and in the life cycle of viral pathogens. There is thus a need for detailed structural and functional information regarding these molecules. A combination of biophysical, chemical, biochemical, and cell biological approaches provides a powerful approach for the analysis of such systems. Modified nucleic acid substrates, synthesized using both chemical and enzymatic methodologies, are very useful probes of the structure and reactions of nucleic acids and nucleic acid-protein complexes.
Specific areas of interest include:
- Mechanisms of pre-mRNA splicing Intron excision from a pre-mRNA substrate involves two sequential transesterification reactions catalyzed by the spliceosome. These reactions are strikingly similar, mechanistically, to those of the self-splicing Group II introns although the evolutionary relationship between the two systems is unclear. It is widely assumed that the catalytic core of the spliceosome is an RNA structure but little is known about the chemical mechanisms of the transesterification reactions or the structure of the substrate bound at the active site. We are addressing these issues by structure/function studies involving chemically modified pre-mRNA splicing substrates and splicing factors. We are also using high resolution structural analysis – X-ray crystallography – to characterize the structures of protein and protein-RNA complexes at the heart of the spliceosome.
- Small RNA-mediated Gene Regulation It has been shown that small RNAs repress or modify gene expression in all organisms. In higher eukaryotes, there are multiple mechanisms including the endogenous miRNA and anti-viral siRNA pathways. We are exploring the CRISPR system a small RNA-dependent acquired immunity in bacteria. Phage resistance is conferred by the specific targeting of foreign nucleic acid by a bacterial surveillance system. We are using biochemical and structural methods to study the mechanism of this process.
Ritchie DB, Schellenberg MJ, Gesner EM, Raithatha SA, Stuart DT, and MacMillan AM. (2008) Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nature Struct. Mol. Biol. 15:1199-1205.
Kent OA and MacMillan AM. Proteins Specifically Modified with a Chemical Nuclease as Probes of RNA-Protein Interaction. in Methods Mol. Biol. 488:191-200 RNA-Protein Interaction Protocols, second edition (R.J. Lin, Ed.; Humana Press, Totowa, N.J., 2008).
Schellenberg MJ, Edwards RA, Ritchie DB, Kent OA, Golas MM, Stark H, Luhrmann R, Glover JNM, MacMillan, A.M. (2006) Crystal Structure of a Core Spliceosomal Protein Interface. Proc. Natl. Acad. Sci. U.S.A. 5:1266-1271.
Kent OA, Ritchie DB, MacMillan AM. E': A Novel Commitment Complex in the Mammalian Spliceosome Assembly Pathway. Mol. Cell. Biol. 25:233-240 (2005).