Cyclic nucleotide-dependent screening of a lamda expression library for nucleic acid binding activities.

Here we describe a modified ligand screening strategy for the expression cloning of mammalian proteins that require the activation of protein kinase cascades to activate ligand binding activity. The manipulation of prokaryotic signaling pathways by the application of appropriate inhibitors or agonists to the nitrocellulose filter during functional screening of standard bacteriophage cDNA expression libraries can permit detection of activities that would not otherwise be found in their active state. We have applied this strategy to a A expression library to clone a novel renal cDNA that exhibits cAMP-dependent RNA binding activity when subsequently tested by ectopic expression in mammalian cells.

HuR binds a cyclic nucleotide-dependent, stabilizing domain in the 3' untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

Differentiation-dependent expression of the Na(+)/glucose cotransporter (SGLT1) is accompanied by a large, cAMP-dependent increase in stability of its mRNA. Stabilization is mediated by protein binding to a critical uridine-rich element (URE) in its 3' untranslated region. In the present study, we demonstrate that HuR, an RNA binding protein of the embryonic lethal abnormal vision family, binds the SGLT1 URE.

A cis-dominant cyclic nucleotide-dependent regulatory domain in the 3'-untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

A 122 nt uridine-rich sequence (URE) in the Na(+)/glucose cotransporter (SGLT1) mRNA 3'-untranslated region is critical for cAMP-dependent message stabilization. Its function was investigated in LLC-PK(1) cells stably expressing beta-globin reporter transcripts. Insertion of the SGLT1 URE downstream from an unrelated destabilizing sequence, the c-fos ARE, evoked cAMP-dependent message stabilization.

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.

Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element.

AU-rich RNA-destabilizing elements (AREs) have become a paradigm for studying cytoplasmic mRNA turnover in mammalian cells. Though many RNA-binding proteins have been shown to bind to AREs in vitro, trans-acting factors that participate in the in vivo destabilization of cytoplasmic RNA by AREs remains unknown. Experiments were performed to investigate the cellular mechanisms and to identify potential trans-acting factors for ARE-directed mRNA decay.