The Structural and Functional Role of Vertebrate LaRP6 N-Terminal Region
Date
2020-05
Authors
Godinez, Brianna
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Abstract
La-related protein 6 (LaRP6) belongs to a superfamily of evolutionarily related RNA-binding proteins. While other LaRP families have been characterized more thoroughly, LaRP6 is still poorly understood. The majority of the work to date has primarily studied the RNA binding domain, the “La Module”, in isolation. However, these studies may not reflect how the full-length protein behaves in vivo. Previous work in our lab using the Danio rerio (zebrafish) LaRP6 protein as a model system demonstrated that a domain adjacent to the La Module, called the “N-terminal region” (NTR), may play an important role in the full-length protein. To study the effect of this domain on the RNA binding activity, we created a new protein construct that contained only the NTR and the La Module while omitting the C-terminal domain, dubbed the “ΔCTD.” The tagged NTR has also been generated, along with the isolated tag to serve as a negative control for biochemical analyses. RNA binding activity was measured using electrophoretic mobility shift assays and showed that, unexpectedly, the ∆CTD exhibited more stable binding as compared to the isolated La Module for all the ligands tested. Both the La Module and ΔCTD bind to the endogenous stem-loop ligand HsCOL1A1 an order of magnitude more tightly than the homopolymers. However, while the ∆CTD binds to Poly-A and Poly-U, no binding was observed to Poly-C, showing that the NTR not only modulates RNA binding activity, but also its ligand specificity. Additionally, the isolated NTR was found to bind independently to HsCOL1A1, albeit weakly. Circular dichroism of the ∆CTD and tagged NTR strongly indicates that the NTR is a random coil.
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Keywords
RNA-binding, LaRP6, Biochemistry, RNA, Protein, Biophysics
Citation
Godinez, B. N. (2020). <i>The structural and functional role of vertebrate LaRP6 N-terminal region</i> (Unpublished thesis). Texas State University, San Marcos, Texas.