Structural characterization of La-Related Protein 6 via cryo-EM and solution-state NMR

Nolan Blackford, Blaine Gordon, Victoria Ogunkunle, Robert Silvers

Florida State University

The La-Related Proteins (LARPs) are a family of RNA binding proteins which have no enzymatic domains but serve a variety of regulatory functions. They all play a role in regulating the Translation of RNAs. These proteins are fascinating because while their RNA binding domains differ only very subtly, the substrates they bind can differ wildly in both structure and sequence. This family of proteins is characterized by the presence of two common domains: the well conserved La-Motif (LaM) and the RNA Recognition Motif (RRM), which varies more between the LARPs than the La-Motif. Both domains have been deemed important in the recognition and direct binding of their target RNAs.

LARP6 upregulates the production of type I collagen, post-transcriptionally, by binding to the untranslated regions of the mRNAs that code the a1 and a2 collagen strands, making it a potential drug target for the treatment of fibrosis. The 5’ UTRs of these mRNAs take on a stem loop secondary structure with a central bulge region which includes the start codon. This structure, as far as it is known, is unique to the a1 and a2 collagen strand’s mRNAs. The structures of LARP6’s La Motif and RRM have been solved as individual domains in the apo form. The structures of the entire La-Module and its domains have yet to be characterized in their bound forms, so LARP6s binding mechanism remains unknown.

While the LARP6 La-Module bound to the collagen stem-loop RNA is theoretically too small for cryo-EM, our strategy is to make the RNA larger by replacing the anticodon loop of a tRNA with the collagen stem-loop RNA recognized by LARP6. The RNA construct we are using is a hybrid between the E. Coli lysine tRNA and the stem loop from the collagen A2 strand’s messenger RNA. Previous attempts to solve small structures via Cryo-EM by attaching them to a larger particle generally fail due to the flexibility of the linker. tRNAs have relatively ridged and stable secondary structures for RNAs and the phosphorus in the backbone provides superior contrast as the heavier nucleus scatters more electrons. Data obtained via cryo-EM will be paired with NMR (nuclear magnetic resonance) data to elucidate both the structure of the RNA binding domains, the role each serves in binding (the role of the RRM is in question as the canonical RNA binding site is obscured in LARP6) as well as their dynamics. Presently, this project is still in progress. Although we are still working on a structure, preliminary data obtained via NMR, microscale thermophoresis, and Negative-Stain EM will be presented. Data obtained from Cryo-EM screening may be presented depending on progress in the next two month.