Biochemical Analysis of Evolutionary Divergent Vertebrate Larp6 Proteins
Date
2017-05
Authors
Castro, Jose M.
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Abstract
The La-related proteins (LARPs) contain a conserved bipartite RNA binding domain, called the La module, which consists of the La motif and an RNA recognition motif (RRM). Across the LARP subfamilies, the La module interacts with a variety of RNAs, allowing this protein superfamily to perform roles as chaperone proteins, post-transcriptional regulatory factors, regulators of RNA metabolism. The ligand specificity of the LARP6 subfamily is still unknown. The only endogenous ligand for LARP6 is found in the 5’ untranslated region of collagen type I mRNA. Animal studies confirmed that the binding of a particular RNA structure by LARP6 was critical for collagen production and muscle development. In this study, we are using the LARP6 proteins from zebrafish (Danio rerio), platyfish (Xiphophorus maculatus) and human to carry out detailed biochemical analyses of vertebrate LARP6 RNA binding activity. The purpose is to determine if there is a species-specific protein-RNA interaction due to co- evolution of the ligand-binding surface within the LARP6 RRM and the RNA ligand sequence and/or structure. Recombinant fish and human LARP6 proteins were expressed in E. coli with an N-terminal SUMO tag. A filtration screening assay was used to identify additives that increase solubility and stability to the fish proteins. To robustly compare direct RNA binding affinity of the fish proteins to the human LARP6, the SUMO tag was cleaved by Ulp1. Limited proteolysis studies were used to compare the domain topology of the fish LARP6 proteins to the human homolog. The RNA binding activity of all LARP6 proteins for the COL1A1 stem loop RNA from the appropriate species was measured by electrophoretic mobility shift assays (EMSAs).
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Keywords
LARP6, Collagen type I mRNA, EMSA
Citation
Castro, J. M. (2017). Biochemical analysis of evolutionary divergent vertebrate Larp6 proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.
Castro, J. M. (2017). <i>Biochemical analysis of evolutionary divergent vertebrate Larp6 proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.
Castro, J. M. (2017). <i>Biochemical analysis of evolutionary divergent vertebrate Larp6 proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.