Rabadan-Diehl Cristina, Volpi Simona, Nikodemova Maria, Aguilera Greti
Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862, USA.
Mol Endocrinol. 2003 Oct;17(10):1959-71. doi: 10.1210/me.2001-0336. Epub 2003 Jul 17.
Posttranscriptional mechanisms play an important role regulating pituitary levels of vasopressin V1b receptors (V1bR) during adaptation to stress. This study investigates the involvement of an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) on V1bR translation. Transfection of bicistronic luciferase constructs into MCF-7 cells showed marked increases in translation of the second cistron after insertion of a 499-bp fragment of the V1bR 5'UTR in the intercistronic region, independently of cap-mediated translation, indicating the presence of IRES activity. IRES-mediated translation was potentiated by the protein kinase C activators, 12-O-tetradecanoylphorbol 13-acetate (PMA) and bryostatin 1, and appears to involve phosphorylation of amino terminus of eIF4G. In Chinese hamster ovary cells transfected with pV1bR-green fluorescent protein (pV1bR-GFP), PMA increased V1bR-GFP protein levels when cap-mediated translation was inhibited by rapamycin. The effect of PMA was due to increased translation because it persisted under transcriptional blockade by actinomycin D, and it was completely abolished by cycloheximide. In addition, PMA stimulated [35S]methionine incorporation into V1bR-GFP but not beta-actin in the absence of mRNA changes. The data show that regulation of IRES activity in the 5'UTR of the V1bR mRNA probably through phosphorylation of eIF4G may serve as a mechanism for rapid changes in V1bR translation to meet physiological demands.
转录后机制在应激适应过程中对垂体血管加压素V1b受体（V1bR）水平的调节起着重要作用。本研究调查了内部核糖体进入位点（IRES）在V1bR翻译的5'非翻译区（5'UTR）中的作用。将双顺反子荧光素酶构建体转染到MCF-7细胞中，结果显示，在顺反子间区域插入V1bR 5'UTR的499 bp片段后，第二个顺反子的翻译显著增加，且与帽介导的翻译无关，这表明存在IRES活性。蛋白激酶C激活剂12-O-十四烷酰佛波醇13-乙酸酯（PMA）和苔藓抑素1可增强IRES介导的翻译，且似乎涉及eIF4G氨基末端的磷酸化。在用pV1bR-绿色荧光蛋白（pV1bR-GFP）转染的中国仓鼠卵巢细胞中，当雷帕霉素抑制帽介导的翻译时，PMA可增加V1bR-GFP蛋白水平。PMA的作用是由于翻译增加，因为在放线菌素D的转录阻断下该作用仍然存在，且被环己酰亚胺完全消除。此外，在mRNA无变化的情况下，PMA刺激[35S]甲硫氨酸掺入V1bR-GFP而非β-肌动蛋白。数据表明，V1bR mRNA的5'UTR中IRES活性的调节可能通过eIF4G的磷酸化，这可能是V1bR翻译快速变化以满足生理需求的一种机制。
