The acute box cis-element in human heavy ferritin mRNA 5'-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins.
Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5'-untranslated region. Iron-responsive element (IRE). Here we show that iron and interleukin-1beta (IL-1beta) act synergistically to increase H- and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5'-untranslated region, conferred a substantial increase in basal and IL-1beta-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-alphaCP1 and -alphaCP2, which have well defined roles as translation regulators. Iron influx increased the association of alphaCP1 with ferritin mRNA and decreased the alphaCP2-ferritin mRNA interaction, whereas IL-1beta reduced the association of alphaCP1 and alphaCP2 with H-ferritin mRNA. In summary, the H-ferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver.
Thomson
Andrew M
AM
Laboratory for Cancer Medicine, School of Medicine and Pharmacology, UWA Centre for Medical Research, Western Australian Institute for Medical Research and University of Western Australia, Royal Perth Hospital.
Cahill
Catherine M
CM
Cho
Hyun-Hee
HH
Kassachau
Kristin D
KD
Epis
Michael R
MR
Bridges
Kenneth R
KR
Leedman
Peter J
PJ
Rogers
Jack T
JT
D020121:5' Untranslated Regions; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D005293:Ferritins / Q000737:chemistry* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007375:Interleukin-1 / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D010957:Plasmids / Q000378:metabolism; D011066:Poly C / Q000737:chemistry; D011257:Pregnancy Proteins / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D020121:5' Untranslated Regions; D007375:Interleukin-1; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011066:Poly C; D005293:Ferritins; D007501:Iron; C454461:FTH1 protein, human; D010088:Oxidoreductases; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase