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首页> 外文期刊>American Journal of Physiology >Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA.
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Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA.

机译：腺苷酸化和AUF1结合在谷氨酰胺酶mRNA的pH响应稳定中的作用。

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During chronic metabolic acidosis, increased expression of renal glutaminase (GA) results from selective stabilization of the GA mRNA. This response is mediated by a direct repeat of an 8-base adenylate-uridylate (AU) sequence that binds zeta-crystallin and functions as a pH response element (pH-RE). A tetracycline-responsive promoter system was developed in LLC-PK(1)-F(+) cells to perform pulse-chase analysis of the turnover of a chimeric beta-globin (betaG) mRNA that contains 960 bp of the 3'-UTR of GA mRNA including the pH-RE. The betaG-GA mRNA exhibits a 14-fold increase in half-life when the LLC-PK(1)-F(+) cells are transferred to acidic medium. RNase H cleavage and Northern blot analysis of the 3'-ends established that rapid deadenylation occurred concomitantly with the rapid decay of the betaG-GA mRNA in cells grown in normal medium. Stabilization of the betaG-GA mRNA in acidic medium is associated with a pronounced decrease in the rate of deadenylation. Mutation of the pH-RE within the betaG-GAmRNA blocked the pH-responsive stabilization, but not the rapid decay, whereas insertion of only a 29-bp segment containing the pH-RE was sufficient to produce both a rapid decay and a pH-responsive stabilization. Various kidney cells express multiple isoforms of AUF1, an AU-binding protein that enhances mRNA turnover. RNA gel-shift assays demonstrated that the recombinant p40 isoform of AUF1 binds to the pH-RE with high affinity and specificity. Thus AUF1 may mediate the rapid turnover of the GA mRNA, whereas increased binding of zeta-crystallin during acidosis may inhibit degradation and result in selective stabilization.
机译：在慢性代谢性酸中毒期间，由于GA mRNA的选择性稳定，导致肾脏谷氨酰胺酶（GA）的表达增加。此响应是由直接结合8碱基腺苷酸-尿苷酸（AU）序列介导的，该序列与zeta-crystallin结合并充当pH响应元件（pH-RE）。在LLC-PK（1）-F（+）细胞中开发了四环素反应性启动子系统，以对包含960 bp的3'-UTR的嵌合β-珠蛋白（betaG）mRNA的更新进行脉冲追踪分析。包括pH-RE的GA mRNA。当LLC-PK（1）-F（+）细胞转移到酸性培养基中时，betaG-GA mRNA的半衰期增加了14倍。 RNase H切割和3'-末端的Northern印迹分析确定，在正常培养基中生长的细胞中，快速的腺苷酸化与betaG-GA mRNA的快速衰减同时发生。在酸性介质中βG-GAmRNA的稳定与腺苷酸化速率的显着降低有关。 betaG-GAmRNA中pH-RE的突变阻止了pH响应的稳定，但不能迅速衰减，而仅插入一个包含pH-RE的29 bp片段足以产生快速衰减和pH-响应稳定。各种肾脏细胞表达AUF1的多种同工型，AUF1是一种可增强mRNA转换的AU结合蛋白。 RNA凝胶位移分析表明，AUF1的重组p40亚型以高亲和力和特异性与pH-RE结合。因此，AUF1可以介导GA mRNA的快速更新，而在酸中毒期间增加的Zeta-crystallin结合可能抑制降解并导致选择性稳定。

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