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Analysis of RNA Binding by Evolutionarily Divergent LARP6 Proteins

The La‐related protein superfamily is known to bind RNAs, and each subfamily appears to bind to distinct kinds of RNAs. The RNA binding activity of La‐related proteins is exerted by a single RNA binding domain, which consists of a well‐conserved La motif (LAM) and a RNA recognition motif (RRM). However, the ligand specificity of the LARP6 subfamily is unknown. Previous research has shown that Homo sapiens LARP6 interacts with the 5′ untranslated region of the mRNA that encodes for collagen type I (COL1A1), specifically recognizing a 48 nt stem‐loop structure. Collagen is an important structural protein that, in muscle tissue, forms part of the endomysium responsible of covering and protecting each individual muscle fiber. Subsequent work found that LARP6/COL1A1 interaction was important for zebrafish embryonic development. Bony fish like zebrafish and platyfish are widely used as animal models and are well‐suited to explore the function and evolution of LARP6 proteins. Zebrafish (Danio rerio) has two LARP6 paralogs (LARP6a and LARP6b), while platyfish (Xiphophorus maculatus) has only one LARP6, were chosen for biochemical analysis of LARP6 RNA binding activity. The sequence alignment of the fish LARP6 proteins compared to human showed significant differences within the RRMs. This study has three specific aims: (1) to determine if XmLARP6 and DrLARP6a bind the respective COL1A1 RNA; (2) to evaluate how species‐specific the LARP6/COL1A1 interaction is by comparing human and fish RNA ligands; and (3) to test if DrLARP6a and DrLARP6b exhibit different RNA binding activities in either affinity or specificity. All LARP6 proteins were expressed as recombinant proteins in E. coli, and purified to >95% purity by nickel affinity and size exclusion chromatography. RNA binding activity is measured by electrophoretic mobility shift assays (EMSAs) using biotinylated RNAs. The fish LARP6 proteins appear to bind their respective COL1A RNA ligands with comparable affinity as the human system. Human LARP6 shows affinity to the fish RNA ligands as well as the fish for the human ones. Differences in RNA binding activity between DrLARP6a and DrLARP6b are expected to provide insight into the subfunctionalization of these two paralogs.