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Studies

Macromolecular Structure Group

Fig 1 - Crystal structure of the bI3 maturase from S. cerevisiae, Fig 2 - Crystal structure of CIRV p19 protein in complex with siRNA, Fig 3 - Crystal structure of HuD RRMs 1 and 2 in complex with AU-rich element RNA, Fig 4 - Crystal structure of the RNA-binding domain of human Pumilio1 protein in complex with target RNA

Understanding RNA Recognition Strategies in Post-Transcriptional Gene Regulation

 

The Macromolecular Structure Group uses molecular, biochemical, and structural approaches to understand how proteins specifically recognize their RNA targets. These studies have revealed some of the diversity of RNA recognition strategies that exist in nature, and they guide our appreciation for how the specificity impacts coordinated regulation of RNA networks.  

 

Examples of studies are below:

 

Post-transcriptional gene regulation by PUF proteins

PUF family proteins are found in organisms from humans to yeast. Typical PUF proteins contain a characteristic sequence-specific RNA-binding domain and regulate gene expression by binding to regulatory sequences and modulating mRNA stability or translation. The Macromolecular Structure Group uses them as a model system for understanding how diversity of RNA recognition properties can be achieved and the correlation between specificity and coordinated gene regulation. In addition, their biological function in stem cell maintenance and cell growth pathways makes their study important to the NIEHS and NIH mission and goals. Crystal structures of different PUF proteins are used to guide functional studies of their RNA recognition properties.

 

Engineering RNA sequence specificity of Pumilio1 protein

Crystal structures of human PUF protein Pumilio1 revealed a modular protein of 8 repeats.  Each repeat binds to an RNA base with two amino acid side chains to specify the recognized base.  Sets of side chains for recognition of RNA bases A, U, and G were deduced from crystal structures and a set for C recognition was determined by selection experiments.  Together this results in a code to design specific recognition of any 8-base sequence using the Pumilio1 scaffold. The group seeks to use this knowledge to design proteins as research tools that can be used to probe cellular function and to direct alternative splicing events critical in human diseases.

 

Mechanism of RNA silencing

Regulation of gene expression by small RNAs, small interfering RNA (siRNA) or microRNA (miRNA), affects most biological pathways with thousands of human genes, perhaps all, targeted by at least one miRNA. Furthermore, RNA interference (RNAi) methods are used routinely in research to knockdown gene expression, small RNA biomarkers of disease and exposure are sought after, and medical applications to modulate gene expression are under development. The Macromolecular Structure Group focuses on understanding the fundamental mechanisms by which these small RNA effector molecules are produced and exert their regulatory effects.

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