Mechanisms of Nuclear Export of the RNA‐Binding Protein LARP6

Abstract

The La Related Protein (LARP) superfamily comprises RNA binding proteins that have diverse RNA targets (including mRNA and tRNA) and exert regulatory functions in the cell. One member of the family, LARP6, regulates the synthesis of type I collagen by binding the 5′ untranslated region of the mRNA and facilitating its localization to the ER membrane. Aberrant collagen expression is a key feature of fibroproliferative diseases such as osteogenesis imperfecta. Therefore, LARP6 is a potential target for treatment of these diseases. However, the mechanisms of LARP6 cellular localization remain unclear. Previously published work transfected a GFP‐tagged LARP6 harboring a deletion of a putative nuclear export sequence (NES‐RBD) within the RNA binding domain. The NES deletion mutant was observed to accumulate in the nucleus, suggesting nucleocytoplasmic shuttling. The purpose of the current study is to understand this nuclear export mechanism in concert with recent structural data for the RNA‐binding domain of LARP6. First, we investigated the localization of endogenous LARP6 in immortalized cell lines using immunocytochemistry and leptomycin B treatment to inhibit classical nuclear transport mechanisms. These experiments demonstrated challenges with the selectivity of commercial antibodies for endogenous levels of human LARP6 in mammalian cells. Additionally, we used a recently‐developed algorithm, LocNES, to identify a second candidate NES motif in the C‐terminus of LARP6 (NES‐CTD) and to clarify the boundaries of the NES‐RBD effectively breaking it into two candidates (NES‐RBD1 and NES‐RBD2). Two approaches were used to test these new candidate motifs. First, the predicted NES motifs were cloned as fusions to GST and purified in preparation for pulldown assays to directly assess interaction with the transporter CRM1. Second, bacterial expression constructs of the wildtype and deletion mutants of the two predicted NES were constructed to analyze the effects of the NES deletions on recombinant protein stability. Chemical and thermal denaturation of the recombinant proteins was monitored by fluorescence spectroscopy to measure the free energy of folding. While all of the GST‐NES fusions are readily expressed in bacteria, only the NES‐CTD candidate motif is readily purified. The high hydrophobicity of the two NES‐RBD candidate motifs appears to prevent their purification. Together, the observed differences in the stability of GST‐NES peptides and the LARP6‐NES deletion mutants suggest that manipulation of the hydrophobic‐rich regions complicates the biochemical efforts to elucidate the mechanism of nuclear export of LARP6.Support or Funding InformationNational Institutes of Health – National Institute of General Medical Sciences (GM119096)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.