van Dijk Erwin, Le Hir Hervé, Séraphin Bertrand
Equipe Labellisée La Ligue, Centre de Génétique Moléculaire, Unité Propre de Recherche 2167, Centre National de la Recherche Scientifique Associée à l'Université Paris VI, Avenue de la Terrasse, 91198 Gif sur Yvette, France.
Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12081-6. doi: 10.1073/pnas.1635192100. Epub 2003 Oct 1.
Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown. In the 3'-5' mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP. In the 5'-3' pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5'-phosphorylated mRNA. We investigated mRNA decay in human cell extracts by using a new assay for decapping. We observed that 5'-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5'-3' mRNA decay pathway. Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated. Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms. The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human. m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay. m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay.
真核生物的mRNA降解通过两条主要途径进行，这两条途径都涉及mRNA帽结构的破坏。在3'-5'mRNA降解途径中，mRNA主体降解产生游离的m7GpppN，其被DcpS水解生成m7GMP。在5'-3'途径中，最近鉴定出的人类Dcp2脱帽酶切割去腺苷酸化mRNA的帽结构，产生m7GDP和5'-磷酸化mRNA。我们通过一种新的脱帽测定法研究了人类细胞提取物中的mRNA降解。我们观察到，在底物RNA与人细胞提取物孵育后，脱帽产生的5'-磷酸化中间体出现，这表明存在活跃的5'-3'mRNA降解途径。然而，令人惊讶的是，未检测到同源的m7GDP产物，而产生了大量的m7GMP。进一步的实验表明，出乎意料的是，m7GDP在来自各种生物体的提取物中能有效地转化为m7GMP。在酵母和人类中，该反应所需且充分的因子被鉴定为DcpS。因此，m7GMP是真核生物mRNA降解的一种普遍的、不依赖途径的副产物。m7GDP的分解应可防止甲基化核苷酸在核酸中的错误掺入，并可能产生一种独特的指示剂，使细胞能够监测mRNA降解。
