Structural and Functional Insights into the RNA-Binding Protein LARP6 using Fish Homologs

Friday, March 22, 2019

Prof. Karen Lewis

Department of Chemistry & Biochemistry

Texas State University

Karen Lewis

All members of the La-Related Protein 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. As these fish represent significant evolutionary divergence from each other and from humans, they also 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. First, limited trypsinolysis and mass spectrometry identified a stable domain in the fish proteins, comprised of the uncharacterized N-terminal domain (NTD) and the La Module. These results indicated that there is direct contact between the NTD and La Module. We are currently employing a suite of biochemical and biophysical approaches to study this intramolecular interaction as a novel mechanism by which the RNA binding activity of LARP6 is regulated. Additionally, the fish La Modules have been amenable to several structural analyses, including solution NMR and small-angle X-ray scattering. In collaboration with the Warner Lab at Boise State, we are using these approaches to determine the relative orientation of the La Motif and RRM within the LARP6 La Module, which is hypothesized to be more extended than what is observed in the La Modules of the nuclear La-related proteins.

Reception: 3:00PM Hand 1135

Seminar: 3:30PM Hand 1144

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