Mechanisms of Human LARP6 Nuclear Export
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The La Related Protein 6 (LARP6) regulates the expression of type I collagen synthesis by binding to the encoding mRNA. However, the molecular mechanism of ligand binding and cellular localization is not fully understood. Understanding the mechanisms of LARP6 regulation requires knowledge of where and how LARP6 functions inside the cell. Previous work suggested that LARP6 undergoes nucleocytoplasmic shuttling, supported by the identification of a putative nuclear export sequence (NES) and to localization in both the nucleus and the cytoplasm. Subsequent high-resolution structures of the RNA binding domain of LARP6 showed that the NES is located in a core part of the RNA Recognition Motif, where it may contribute to RNA binding and/or be a core structural element. The goals of this thesis were to combine these cellular and structural models of LARP6 localization into a cohesive model of LARP6 function. First, to test if LARP6 export is mediated by the nuclear export in Chromosome Region Maintenance 1(CRM1) in vivo, we performed inhibition studies in cell lines that endogenously expressed LARP6. Second, we developed two related approaches that leveraged the recombinant LARP6 protein approaches that had been established in the lab. To determine whether computationally-identified sequences are responsible for nuclear export, we recombinantly expressed putative NES motifs as glutathione S-transferase (GST) fusion proteins for use in in vitro CRM1 pulldown assays. We identified three additional putative NES using LocNES, a more recent and robust NES identification algorithm. We have also designed NES deletion mutants, which will be used to examine the effects on structural stability caused by the deletion of all putative NES motifs by introducing those mutations into the recombinant, full-length protein construct that is expressed in E. coli.