Regulation of insulin-like growth factor binding protein-5 mRNA abundance in rat intestinal smooth muscle.
IGF-I increases abundance of IGFBP-5 mRNA in rat intestinal smooth muscle cells (RISM), and IGFBP-5 protein in RISM conditioned media. The translational blocker, cycloheximide, decreased the abundance of IGFBP-5 mRNA to undetectable levels, suggesting that IGFBP-5 mRNA integrity is linked to protein synthesis. We studied the mechanism of IGF-I's effect on IGFBP-5 mRNA, and the role of cytoplasmic proteins in modulating IGFBP-5 mRNA abundance. Anisomycin, emetine, and puromycin abolished IGFBP-5 mRNA as seen with cycloheximide. Cycloheximide had a dose- and time-dependent effect on IGFBP-5 mRNA. IGF-I increased IGFBP-5 nuclear transcripts by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that IGF-I acts at least partially by increasing IGFBP-5 mRNA transcription. Protein synthesis inhibitors did not affect IGFBP-5 nuclear transcripts, therefore, they affect only mature mRNA. The IGFBP-5 mRNA 3' and 5' UTRs were cloned and their sequences searched for adenosine-uridine rich elements (AUREs), elements shown to regulate RNA stability. RNA mobility gel shift assay showed two protein activities that bind to nt 922 to 2076 of the 3' UTR, a region that contains an AURE. One protein activity (BA2) was decreased in cytoplasmic extracts from cycloheximide-treated RISM. These data demonstrate that IGFBP-5 mRNA integrity is dependent on protein synthesis. The 3' UTR of IGFBP-5 contains elements shown to bind proteins important for RNA stability regulation. This region binds RISM cytoplasmic proteins, and may mediate the dramatic effect of cycloheximide on IGFBP-5 abundance. RNA-protein interactions may be important to IGFBP-5 mRNA stability and ultimately, to IGFBP-5 actions.
Hou
Y T
YT
Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-0589, USA.
Xin
X P
XP
Li
L
L
Zimmerman
E M
EM
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D009130:Muscle, Smooth / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D007334:Insulin-Like Growth Factor I; D003513:Cycloheximide