Effect of the N‐terminal Region of Arabidopsis thaliana LARP6C in RNA Binding Specificity

Abstract

The La‐related proteins (LARPs) are a superfamily of RNA‐binding proteins that are distinguished by a core RNA binding domain called the “La Module”. This family is highly conserved across eukaryotes and exerts diverse functions in RNA processing and function. The genetic model vascular plant, Arabidopsis thaliana (At), has three paralogs of LARP6, denoted “A”, “B”, and “C”. Of these paralogs, AtLARP6B and AtLARP6C are more closely related to each other due to an N‐terminal sequence motif known to associate with other RNA binding proteins, the “PAM2w” motif. Previous work evaluated the RNA binding activity of the isolated AtLARP6C La Module. Recent work on vertebrate LARP4A suggests that N‐terminal PAM2 motifs are important for higher‐order assembly of regulatory complexes. We have generated a set of recombinant constructs to test the role of both the NTR and PAM2w motif in RNA binding activity. These protein variants are stably expressed and have been highly purified for use in electrophoretic mobility shift assays (EMSAs) to measure RNA binding activity. Preliminary work indicates that binding affinity is not significantly impacted by either the NTR or the PAM2w motif, and current studies are focusing on the role that these regions play in ligand specificity. By characterizing the binding specificity of these AtLARP6C proteins to a panel of RNA ligands, we will gain significant insight into the contribution of this domain and motif to ligand binding.