mRNA localization in polarized intestinal epithelial cells.
An important feature of enterocyte maturation is the asymmetrical distribution of cellular functions including protein localization. mRNA sorting is one mechanism for establishment and maintenance of this process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2 cells. Proxy cDNA constructs containing beta-galactosidase (beta-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A sequence in place of 3'-UTR. Expression of GFP was observed by confocal microscopy; intracellular location of the construct mRNA was imaged by in situ hybridization. The sucrase-isomaltase mRNA proxy localized to an apical position in Caco-2 cells as in native enterocytes; the villin mRNA proxy did not show significant localization. The control construct was not localized and was found diffusely throughout the cell. Proxy GFP proteins tended to localize with their mRNA proxies, but with less precision. This study establishes a valuable model for the investigation of mRNA localization in intestinal epithelial cells. Mechanisms controlling asymmetrical distribution of intestinal mRNAs can be now be elucidated.
Houle
Vicki M
VM
Gastrointestinal Cell Biology Research, Division of Gastroenterology and Nutrition, Harvard Digestive Disease Center, Children's Hospital, Harvard Medical School, Boston 02115, USA.
Li
Wei
W
Montgomery
Robert K
RK
Grand
Richard J
RJ
D020413:3' Untranslated Regions / Q000235:genetics; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics; D016764:Cell Polarity / Q000502:physiology; D020895:Enterocytes / Q000166:cytology* / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology* / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics; D008840:Microfilament Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013394:Sucrase-Isomaltase Complex / Q000235:genetics*; D014162:Transfection
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D012333:RNA, Messenger; C026615:villin; D049452:Green Fluorescent Proteins; D013394:Sucrase-Isomaltase Complex