Regulation of c-myc mRNA decay by translational pausing in a coding region instability determinant
Regulation of c-myc mRNA decay by translational pausing in a coding region instability determinant
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DOI：
10.1128/mcb.22.12.3959-3969.2002
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发表时间：
2002-06-01
影响因子：
5.3
通讯作者：
Ross, J
中科院分区：
文献类型：
--
作者：
Lemm, I;Ross, J
249个核苷酸的编码区不稳定决定簇(CRD)破坏了c-myc mRNA的稳定性。以前的实验发现了一种CRD结合蛋白(CRD-BP)，它似乎可以保护CRD免受核酸内切酶的切割。然而，目前还不清楚为什么需要CRD-BP来保护一个编码区覆盖着核糖体的翻译良好的mRNA。我们假设CRD中的翻译暂停会在暂停位点下游产生一个核糖体缺陷区，如果不被CRD-BP保护，该区域就会受到内切酶的攻击。本文报道的转染法和无细胞转化法实验支持这一假设。在tRNA耗竭的网织红细胞翻译反应中，核糖体暂停发生在c-myc CRD中。停顿位点映射到稀有精氨酸(CGA)密码子和邻近的苏氨酸(ACA)密码子。将这些密码子改变为更常见的密码子可以提高体外翻译效率，并增加转基因细胞中的mRNA丰度。这些数据表明，c-myc mRNA迅速降解，除非(I)在没有停顿的情况下翻译或(Ii)在停顿发生时受到CRD-BP的保护。进一步的作图实验表明，CRD是两部分的，上游有几个翻译暂停位点，下游有一个核酸内切酶切割位点。
A 249-nucleotide coding region instability determinant (CRD) destabilizes c-myc mRNA. Previous experiments identified a CRD-binding protein (CRD-BP) that appears to protect the CRD from endonuclease cleavage. However, it was unclear why a CRD-BP is required to protect a well-translated mRNA whose coding region is covered with ribosomes. We hypothesized that translational pausing in the CRD generates a ribosome-deficient region downstream of the pause site, and this region is exposed to endonuclease attack unless it is shielded by the CRD-BP. Transfection and cell-free translation experiments reported here support this hypothesis. Ribosome pausing occurs within the c-myc CRD in tRNA-depleted reticulocyte translation reactions. The pause sites map to a rare arginine (CGA) codon and to an adjacent threonine (ACA) codon. Changing these codons to more common codons increases translational efficiency in vitro and increases mRNA abundance in transfected cells. These data suggest that c-myc mRNA is rapidly degraded unless it is (i) translated without pausing or (ii) protected by the CRD-BP when pausing occurs. Additional mapping experiments suggest that the CRD is bipartite, with several upstream translation pause sites and a downstream endonuclease cleavage site.