DISTINCT mRNA DISTRIBUTION PATTERNS IN HUMAN JEJUNAL ENTEROCYTES.

Abstract

70 Although intracellular sorting of mRNAs has been demonstrated in several cellular systems, including oocytes, embryos, neurons, fibroblasts, and absorptive enterocytes, its importance in enterocyte differentiation is poorly understood. Previous studies from our laboratory have demonstrated that mRNAs are asymmetrically distributed in human intestinal epithelial cells, but in general co-localized with their encoded proteins. Most mRNA sorting studies indicate that cis-acting sequences responsible for mRNA localization are located in the 3′-untranslated region (3′UTR). The aims of this study were to characterize in human enterocytes the mRNA localization patterns of genes with distinctly different functions: villin, brush border myosin I (BBMI), Na+-dependent glucose co-transporter (SGLT1), and liver fatty acid binding protein (L-FABP), and to characterize the protein(s) that may be involved in the mRNA sorting process. Methods: mRNA distribution in formalin fixed human jejunal tissue sections was determined byin situ hybridization at high stringency using digoxigenin-labeled RNA probes. Gel-shift assays were used to study RNA-protein interactions in the 3′UTR. Results: The mRNAs for villin and BBMI, well-characterized apical microvillus cytoskeletal proteins, were unexpectedly sorted to the basal region of enterocytes, a different intracellular location than their encoded proteins. The mRNA for human SGLT1 was sorted to the apical region, a location near its encoded protein; and the mRNA for human L-FABP was distributed diffusely in the cytoplasm as is its encoded protein. Gel-shift assays indicated that a cytoplasmic protein in Caco-2 cells binds to the villin 3′UTR, suggesting that this region contains a protein-binding motif. Conclusions: This study not only demonstrates distinct mRNA sorting patterns in human absorptive enterocytes for villin, BBMI, SGLT1, and L-FABP, but also reveals for the first time that a dichotomy may exist between the distribution pattern of mRNA and that of its sorted protein. The observations further offer the opportunity to identify the mechanisms by which mRNAs are separately localized to apical and basal domains in absorptive enterocytes.