Projects working on:

1) Altering the specificity of RNA-binding proteins by in vitro selection and computational design RNA-binding proteins (RBPs) perform essential functions in the post-transcriptional control of gene expression and therefore are fundamental to basic cellular physiology and to many human diseases. These proteins recognize RNA by using relatively few RNA-binding modules that combine to create versatile binding surface to define their specificity. If we were able to engineer RBPs with new RNA-binding specificity, it would become possible to develop new tools to control and investigate gene expression pathways, much like re-engineered zinc-finger proteins that are now being used to control transcription. I have been developing both computational approaches (1) and molecular evolution methods such as in vitro compartmentalization (2) to achieve this goal. Several protein-RNA complexes are currently chosen as testing sets to redirect their RNA-binding specificity.

• 

• Schematic representation of the strategy used in selection of RBPs by IVC

2) NMR structural studies of telomerase RNA Telomeres are DNA-protein complexes that cap the end of linear chromosomes and enhance their genetic stability by protecting DNA ends from recombination, degradation and fusion with other chromosomes. The natural loss of telomeres that occurs during normal cell division is counteracted by the de novo addition of telomeric repeats carried out by telomerase. This enzyme has two core components: a protein (TERT) and an RNA (TER). Conserved domains of telomerase RNA have been shown to be important for efficient telomerase activity and processivity. I have studied one of those regions (Tetrahymena thermophila stem-loop IV) as part of a broad effort in the group to probe the structure-function relationship of telomerase RNA. In vitro transcription method was used to prepare the labeled and unlabelled RNA fragments. Various NMR methods and SHAPE assay have been carried out to determine the structure (3).

• 

• Secondary structure of Tetrahymena telomerase RNA and the NMR structure of stem-loop IV

References:

1. Y. Chen, J. Mandic, G. Varani (2008) Cell-free selection of RNA-binding proteins using in vitro compartmentalization. Nucleic Acids Res. 36, e128.
2. Y. Chen, T. Kortemme, T. Robertson, D. Baker, G. Varani (2004) A new hydrogen bonding potential for the design of protein-RNA interactions predicts specific contacts and discriminates decoys. Nucleic Acids Res. 32, 5147-5162.
3. Y. Chen, J. Fender, B. M. Tracy, G. Varani (2006) Structure of stem-loop IV of Tetrahymena telomerase RNA. EMBO J. 25, 3156-3166.