Cyclic nucleotide regulation of Na+/glucose cotransporter (SGLT1) mRNA stability. Interaction of a nucleocytoplasmic protein with a regulatory domain in the 3'-untranslated region critical for stabilization.
Expression of the Na(+)-coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylation-dependent binding of cytoplasmic proteins to a uridine-rich sequence (URE) in the 3'-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inserting it into the 3'-UTR of a beta-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the beta-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the beta-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase A-dependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridine-rich sequence in the 3'-UTR as critical for cAMP-mediated SGLT1 message stabilization.
Lee
W Y
WY
Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, Texas 77225, USA.
Loflin
P
P
Clancey
C J
CJ
Peng
H
H
Lever
J E
JE
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D020413:3' Untranslated Regions / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry*; D051269:Sodium-Glucose Transporter 1; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020413:3' Untranslated Regions; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D051269:Sodium-Glucose Transporter 1; D012313:RNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine