Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs.
Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs.
复制标题
受精海胆卵中信使 RNA 和鸟嘌呤核苷酸交换因子同时去抑制的证据。
DOI：
10.1016/0012-1606(87)90394-0
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发表时间：
1987
影响因子：
2.7
通讯作者：
Hille,MB
中科院分区：
文献类型：
--
作者：
Colin,AM;Brown,BD;Dholakia,JN;Woodley,CL;Wahba,AJ;Hille,MB
在 Lytechinus pictuse 卵和受精卵提取物中研究了翻译控制。我们表明，mRNA 和起始因子都不能单独限制这些裂解物中的翻译；相反，它们一起限制速率。添加的珠蛋白 mRNA 在卵子和受精卵裂解物中进行翻译，但由于添加的 RNA 与内源信息竞争，总体蛋白质合成并未显着增加。由于将球蛋白 mRNA 显微注射到 L 中，裂解物模拟了体内反应。 pictuseggs 同样与内源 mRNA 竞争。使用许多翻译成分来确定它们是否会刺激这些裂解物中的蛋白质合成。球蛋白多聚核糖体的添加增加了蛋白质合成的水平。这种增加的大部分是由于球蛋白 mRNA 的重新启动，并且在这些条件下，卵子和受精卵提取物中内源蛋白质合成的水平没有变化。单独添加粗起始因子并没有明显改变卵裂解物中蛋白质合成的速率。然而，在添加 mRNA 的情况下，这些起始因子将翻译刺激两到四倍。在所有测试的起始因子中，只有鸟嘌呤核苷酸交换因子（GEF、eIF-2B、RF）在存在珠蛋白 mRNA 时显着增加蛋白质合成。在添加 GEF 和 mRNA 的情况下，添加未分级的起始因子制剂进一步刺激了蛋白质合成，表明除 mRNA 和 GEF 之外的成分在这些卵裂解物中也受到限制。其他起始因子，包括 eIF-2、eIF-4A、eIF-4B 和 eIF-4F，不能替代普通起始因子制剂中的成分。我们认为，细胞质的碱化以及随后启动因子和 mRNA 的激活有助于海胆卵受精后蛋白质合成的大量刺激。此外，我们讨论了受精后 3 分钟内发生的以 NAD+ 为代价的 NADPH 增加可能导致 GEF 激活的可能性。
Translational control was studied in extracts ofLytechinus pictuseggs and zygotes. We showed that neither mRNA nor initiation factors alone limit translation in these lysates; rather they are together rate limiting. Added globin mRNA was translated in egg and zygote lysates but overall protein synthesis did not increase significantly as the added RNA competed with the endogenous message. The lysates mimicked thein vivoresponse, since microinjection of globin mRNA intoL. pictuseggs similarly competed with endogenous mRNAs. A number of translational components were used to determine if they would stimulate protein synthesis in these lysates. The addition of globin polyribosomes increased the level of protein synthesis. The majority of this increase was due to reinitiation of the globin mRNA, and under these conditions the level of endogenous protein synthesis in both egg and zygote extracts did not change. The addition of crude initiation factors alone did not appreciably alter the rate of protein synthesis in the egg lysates. However, in the presence of added mRNA, these initiation factors stimulated translation two- to fourfold. Of all the initiation factors tested, only the guanine nucleotide exchange factor (GEF, eIF-2B, RF) significantly increased protein synthesis when globin mRNA was present. The addition of an unfractionated initiation factor preparation further stimulated protein synthesis in the presence of added GEF and mRNA, suggesting that a component other than mRNA and GEF was also limiting in these egg lysates. Other initiation factors, including eIF-2, eIF-4A, eIF-4B, and eIF-4F, did not substitute for the component in the unfractionated initiation factor preparation. We propose that alkalinization of the cytoplasm and the subsequent activation of initiation factors and mRNAs contribute to the large stimulation of protein synthesis in echinoid eggs after fertilization. Furthermore, we discuss the possibility that the increase in NADPH at the expense of NAD+, which occurs within 3 min after fertilization, may lead to the activation of GEF.