Structural and functional characterization of the human Nuclear Exosome Targeting complex

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Sean Johnson


The human Nuclear Exosome Targeting (NEXT) is a three-protein complex which plays a central function in RNA surveillance and decay in the nucleus of human cells. The NEXT complex is composed of an ATP-dependent RNA helicase (hMtr4), a putative Zn2+ knuckle containing protein (Zcchc8), and a DNA/RNA binding protein (RBM7). The objective of this research is to understand the function of the NEXT complex through structural and biochemical characterization of the individual components of this complex. An overarching goal in this research is to not only understand the individual roles of these proteins, but also to understand how they interact with one another to perform multiple functions. To this end, all three proteins have been cloned into E. coli expression vectors. Truncation mutants and fusion constructs for these proteins have been developed to improve protein stability, solubility and facilitate purification. Robust expression and purification protocols have been established for hMtr4 and RBM7. Biochemical characterization of these proteins is currently underway. Preliminary results suggest differences in the ATPase activity of hMtr4 and its yeast homolog. For RBM7, sequence analysis reveals that this protein contains and RNA Recognition Motif (RRM), which often binds RNA or DNA. We are beginning to outline the RNA/DNA binding preferences of the RRM from RBM7 using EMSA analysis. Preliminary EMSA analyses on the RRM of RBM7 demonstrate that this protein fragment can bind RNA and DNA substrates. Crystallization trials are currently underway to further characterize the RNA binding interface of RBM7.

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