Differential inhibition of mRNA degradation pathways by novel cap analogs
Differential inhibition of mRNA degradation pathways by novel cap analogs
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DOI：
10.1074/jbc.m509121200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
--
作者：
Grudzien, E;Kalek, M;Rhoads, RE
mRNA降解主要通过两种替代途径进行：5' -> 3'途径，其需要去腺苷化，然后脱帽和5' -> 3' ->水解;和3' -> 5'途径，其涉及去腺苷化，然后3' -> 3'水解，最后脱帽。每种途径的机制和相对贡献尚未完全了解。我们研究了不同帽结构（Gp（3）G、m（7）Gp（3）G或m（2）（7，3- O）Gp（3）G）和3'末端（A（31）、A（60）或G（16））对哺乳动物细胞中mRNA翻译和降解的影响。结果表明，以更高亲和力结合eIF 4 E的帽结构使mRNA稳定以在体内降解。mRNA的稳定性取决于5'端结合eIF 4 E的能力，而不仅仅是5'端阻断基团的存在。在5 '-UTR中引入茎环，显著减少翻译，但保持帽结构相同，不会改变mRNA降解的速率。为了测试5' -> 3'与3' -> 5'途径的相对贡献，我们设计并合成了两种新的帽类似物，其中亚甲基在α-和β-磷酸部分之间被取代，m（2）（7，3-O）GppCH（2）pG和m（2）（7，3-O）GpCH（2）ppG，它们被预测对Dcp 1/Dcp 2和DcpS的切割具有抗性，分别这些帽类似物被eIF 4 E识别，并在体外和体内赋予帽依赖性翻译为mRNA。用m（2）（7，3-O）GppCH（2）pG封端的寡核苷酸在体外对重组人Dcp 2的水解具有抗性。用m（2）（7，3-O）GppCH（2）pG而不是m（2）（7，3-O）GpCH（2）ppG封端的mRNA在体内更稳定，表明5 '-> 3'途径对总体降解做出主要贡献。在所有测试的mRNA中，含有5 '-末端m（2）（7，3-O）GppCH（2）pG和3'-末端poly（G）的荧光素酶mRNA具有最大的稳定性。
mRNA degradation predominantly proceeds through two alternative routes: the 5' -> 3' pathway, which requires deadenylation followed by decapping and 5' -> 3' -> hydrolysis; and the 3' -> 5' pathway, which involves deadenylation followed by 3' -> 3' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2) (7,3- O)Gp(3)G) and 3' termini (A(31), A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'- end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5' -> 3' versus 3' -> 5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta- phosphate moieties, m(2) (7,3-O)GppCH(2)pG and m(2) (7,3-O)GpCH(2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2) (7,3-O)GppCH(2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2) (7,3-O)GppCH(2)pG, but not m(2)(7,3-O)GpCH(2)ppG, were more stable in vivo, indicating that the 5'-> 3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2) (7,3-O)GppCH(2)pG and 3'- terminal poly(G) had the greatest stability of all mRNAs tested.