The Structural and Functional Role of Vertebrate LARP6 N‐Terminal Region

Abstract

La‐related protein 6 (LARP6) belongs to a superfamily of evolutionarily related RNA‐binding proteins. While other LARP families have been characterized more thoroughly, LARP6 is still poorly understood. The majority of the work to date for LARP6 focuses on its RNA binding activity, and has primarily studied the RNA binding domain, the “La Module”, in isolation. However, these studies may not reflect how the full‐length protein behaves in vivo. Previous work in our lab using the Danio rerio (zebrafish) LARP6 protein as a model system demonstrated that a domain adjacent to the La Module, called the “N‐terminal region” (NTR), may play an important role in the full‐length protein. To study the effect of this domain on the RNA binding activity, we created a new protein construct that contained only the NTR and the La Module while omitting the C‐terminal domain, dubbed the “ΔCTD.” The NTR has also been created with an attached His10‐SUMO tag, with the additional creation of the isolated tag to serve as a negative control for biochemical analyses. Several features have been compared between the La Module and our ΔCTD construct, with supporting evidence from the NTR. First, size exclusion chromatography and analytical ultracentrifugation demonstrated that, unlike the La Module, the ΔCTD forms dimers under nonreducing conditions. These dimers were resolved by the addition of reducing agent, suggesting that the dimerization was caused by a disulfide bond. RNA binding activity was measured using electrophoretic mobility shift assays and showed that, unexpectedly, the ΔCTD exhibited distinct binding behavior as compared to the isolated La Module. The ΔCTD appears to exhibit increased affinity to homopolymers poly‐A, poly‐U, and the endogenous stem‐loop ligand HsCOL1A1, but no binding was observed to poly‐C, showing that the NTR not only modulates the RNA binding activity, but also its ligand specificity. Circular dichroism of the ΔCTD and SUMO‐tagged NTR strongly indicates that the NTR is a random coil. Small‐angle X‐ray scattering (SAXS) analysis supports that the NTR is able to interact with the La Module to modulate RNA binding behavior.Support or Funding InformationNational Institutes of Health – National Institute of General Medical Sciences (R15GM119096), National Institute of Health project ALS‐ENABLE (P30 GM124169) and a High‐End Instrumentation Grant (S10OD018483)