Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein L
Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein L
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DOI：
10.1074/jbc.274.3.1359
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发表时间：
1999-01-15
影响因子：
4.8
通讯作者：
Claffey, KP
中科院分区：
文献类型：
--
作者：
Shih, SC;Claffey, KP
人血管内皮生长因子（VEGF）3 '非翻译区的一个126碱基的区域被我们鉴定为缺氧稳定区，在RNA-UV-crosslinking分析中形成7个缺氧诱导的RNA-蛋白复合物，其表观分子量范围为40 - 90 kDa。在这项研究中，我们发现，蛋白质，形成60 kDa的RNA-蛋白质复合物与缺氧稳定区存在于细胞质和核室。我们纯化的蛋白质相关的60 kDa的复合物，并确定其为异质核核糖核蛋白L（hnRNP L）的蛋白质测序。通过免疫沉淀去除hnRNP L特异性地废除了60-kDa复合物的形成。合成的脱氧核苷酸竞争性研究确定hnRNPL的RNA结合位点为21个碱基长的序列，5 '-CACCCACCCA-ACA-3'。hnRNP L的免疫沉淀，然后通过逆转录-聚合酶链反应表明，hnRNP L特异性相互作用的VEGF mRNA在缺氧细胞在体内。此外，当用反义寡脱氧核苷酸转染M21细胞的hnRNP L RNA结合位点时，VEGF mRNA的半衰期在缺氧条件下显著降低。因此，我们建议，特定协会的hnRNP L与VEGF mRNA缺氧可能发挥重要作用，在缺氧诱导的转录后调节VEGF mRNA的表达。
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 th, RNA-binding site of hnRNP L as a 21-base-long sequence, 5'-CACCCACCCA-CAUACAUACA-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 posttranscriptional regulation of VEGF mRNA expression.