Dr. Karen Lewis on Structural and functional insights from a fish homolog of the RNA-binding protein LaRP6

University of Pittsburgh Department of Biological Sciences presents:
2019 Fall Seminar Series 

Dr. Karen Lewis
Texas State University 

"Structural and functional insights from a fish homolog of the RNA-binding protein LaRP6"

All members of the La-Related Protein (LaRP) superfamily use an RNA Recognition Motif (RRM) in tandem with a conserved La Motif to bind RNA ligands. However, LaRP6 has evolved unique structural and functional characteristics within the La Module that distinguish it from other LARP subfamilies. In particular, the La Motif—RRM interdomain linker and the RNA binding surface in the RRM appear to be specific to the LARP6 subfamily. To identify critical sequences and motifs that are involved in the structure and function of LARP6, we have employed a comparative phylogenetic approach using the LARP6 proteins from two teleost fish, Danio rerio and Xiphophorus maculatus. These fish represent significant evolutionary divergence from each other and from humans, and provide a natural suite of sequence variants within regions of interest in the RRM. Using an iterative, filter-based screening assay, we successfully purified biochemical quantities of the full-length fish LaRP6 proteins. Electrophoretic mobility shift assays demonstrated that these non-mammalian vertebrate LaRP6 proteins exerted robust RNA binding activity. The fish proteins have provided a system in which to evaluate several features of LaRP6 structure. We identified a stable domain in the fish proteins, comprised of the uncharacterized N-terminal domain (NTR) and the La Module. This domain is collectively termed the “N-terminal domain” (NTD). Despite retaining no detectable secondary structure when in the NTD construct, the NTR was found to modulate the RNA binding activity of the La Module. In particular, the presence of the NTR reduces the binding affinity for the LaRP6 cognate ligand, a stem-loop structure from collagen mRNAs. Remarkably, the NTR increases binding affinity for polyA and polyU ligands. Preliminary small-angle X-ray scattering data indicate that the disordered NTR is likely interacting with the La Module. In collaboration with the Warner Lab at Boise State, we are using these and other biophysical approaches to determine the relative orientation and interaction between the NTR, the La Motif, and the RRM to understand the molecular mechanism of RNA binding by vertebrate LaRP6.

Monday, October 21, 2019
169 Crawford Hall

11:00 A.M.
10:50 A.M.  refreshments 

Host: Dr. Andrea Berman 


21 Oct 2019
Departmental Seminars

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