Identification of Major Boundaries for Structural Characterization of the LARP6 Proteins From Fish
Authors: Pena Eliana Lee , Külköylüoglu Hatice , Castro Jose Miguel , Pu Xinzhu , Warner Lisa R. , Lewis Karen A. 
Published: June 2021

La‐related protein 6 (LARP6) is a posttranscriptional regulator of collagen type I expression in vertebrates. It binds a conserved stem loop structure in the untranslated region of the COL1A1 mRNA. LARP6 contains an uncharacterized N‐terminal domain, a centrally‐located RNA binding domain, and an uncharacterized C‐terminal domain that includes a proline‐rich motif called the LSA motif. The human LARP6 La Module has been structurally characterized as two subdomains: a winged‐helix La Motif (LaM) and an RNA recognition motif (RRM). Both domains are necessary to carry out the RNA binding activity. Previous work in the lab identified that the LARP6 proteins from two bony fish, Danio rerio and Xiphophorus maculatus, contain a proteolytically‐resistant structure that is comprised of both the La Module and the uncharacterized N‐terminal domain. To better understand the interactions between these two components, we set out to precisely identify the domain boundaries of the N‐terminal domain (NTD), La Module (LaMod), and C‐terminal domain (CTD) in the fish LARP6 proteins. We predicted domain boundaries based on multiple sequence alignments, and cloned those boundaries into bacterial expression vectors to create constructs for the isolated La Module and a deletion of just the C‐terminal domain (DCTD = NTD+LaMod) for Homo sapiens, Danio rerio, and Xiphophorus maculatus LARP6. All six of these proteins have been recombinantly expressed and purified using immobilized affinity and size exclusion chromatography. The purified proteins were screened for proper folding by analyzing RNA binding activity using electrophoretic mobility shift assay with the HsCOL1A1 RNA ligand. Proteins were also analyzed for structural stability using limited proteolysis with either trypsin or aminopeptidase. The proteolysis reactions were evaluated by SDS‐PAGE and silver stain, and the major bands excised and further analyzed by mass spectrometry. By mapping the recovered proteolytic peptides onto the protein sequence, we were able to identify conservative boundaries for each of the constructs, which were then tested by the construction, purification, and biochemical analysis of new recombinant protein constructs. Having precise boundaries for the stable domains within the fish LARP6 proteins will enable the structural characterization of these orthologs for comparison to the human protein. This future work will provide insight into the variation of structure and function of these highly‐conserved post‐transcriptional regulators.Support or Funding InformationFunding provided by the Texas State University Student Undergraduate Research Fund (to E.L.P.), the National Institutes of Health – National Institute of General Medical Studies (GM119096 to K.A.L; GM102783, the South Texas Doctoral Bridge Program, supporting J.M.C., E.S. & E.L.P., and GM107759).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.