Novel function of the poly(C)-binding protein alpha CP3 as a transcriptional repressor of the mu opioid receptor gene.
The alpha-complex proteins (alphaCP) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). These proteins are mainly involved in various post-transcriptional regulations (e.g., mRNA stabilization or translational activation/silencing). Here we report a novel function of alphaCP3, a member of the alphaCP family. alphaCP3 bound to the double-stranded poly(C) element essential for the mu opioid receptor (MOR) promoter and repressed the promoter activity at the transcriptional level. We identified alphaCP3 using affinity column chromatography containing the double-stranded poly(C) element and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. AlphaCP3 binding to the poly(C) sequence of the MOR gene was sequence specific, as confirmed by the supershift assay. In cotransfection studies, alphaCP3 repressed the MOR promoter only when the poly(C) sequence was intact. Ectopic expression of alphaCP3 led to repression of the endogenous MOR transcripts in NS20Y cells. When alphaCP3 was disrupted using small interfering RNA (siRNA) in NS20Y cells, the transcription of the endogenous target MOR gene was increased significantly. Our data suggest that alphaCP3 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of alphaCP3 as a transcriptional repressor in MOR gene regulation.
Choi
Hack Sun
HS
Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. choix074@umn.edu
Kim
Chun Sung
CS
Hwang
Cheol Kyu
CK
Song
Kyu Young
KY
Law
Ping-Yee
PY
Wei
Li-Na
LN
Loh
Horace H
HH
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D011066:Poly C / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C525095:Pcbp3 protein, mouse; D016601:RNA-Binding Proteins; D017450:Receptors, Opioid, mu; D012097:Repressor Proteins; D011066:Poly C