Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein L.
A 126-base region of human vascular endothelial growth factor (VEGF) 3'-untranslated region, which we identified as the hypoxia stability region, forms seven hypoxia-inducible RNA-protein complexes with apparent molecular masses ranging from 40 to 90 kDa in RNA-UV-cross-linking assays. In this study, we show that proteins that form the 60-kDa RNA-protein complex with the hypoxia stability region were present in both cytoplasmic and nuclear compartments. We purified the protein associated in the 60-kDa complex and identified it as heterogeneous nuclear ribonucleoprotein L (hnRNP L) by protein sequencing. Removal of hnRNP L by immunoprecipitation specifically abolished formation of the 60-kDa complex. Synthetic deoxyribonucleotide competition studies defined the RNA-binding site of hnRNP L as a 21-base-long sequence, 5'-CACCCACCCACAUACAUACAU-3'. Immunoprecipitation of hnRNP L followed by reverse transcription-polymerase chain reaction showed that hnRNP L specifically interacts with VEGF mRNA in hypoxic cells in vivo. Furthermore, when M21 cells transfected with antisense oligodeoxyribonucleotide to the hnRNP L RNA-binding site, the VEGF mRNA half-life was significantly reduced under hypoxic conditions. Thus, we propose that specific association of hnRNP L with VEGF mRNA under hypoxia may play an important role in hypoxia-induced post-transcriptional regulation of VEGF mRNA expression.
Shih
S C
SC
Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Claffey
K P
KP
D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000235:genetics*; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D008222:Lymphokines / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008222:Lymphokines; D020319:Oligodeoxyribonucleotides, Antisense; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors