Kshirsagar Meenakshi, Parker Roy
Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson 85721-0106, USA.
Genetics. 2004 Feb;166(2):729-39. doi: 10.1534/genetics.166.2.729.
The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.
酵母中mRNA降解的主要途径始于去腺苷酸化，随后是mRNA脱帽和5'-3'核酸外切酶消化。采用计算机方法来鉴定参与mRNA降解途径的新蛋白质。其中一种蛋白质Edc3p，被鉴定为一种功能未知的保守蛋白质，它与几种参与mRNA脱帽和5'-3'降解的蛋白质存在广泛的双杂交相互作用，这些蛋白质包括Dcp1p、Dcp2p、Dhh1p、Lsm1p以及5'-3'核酸外切酶Xrn1p。我们发现，当脱帽酶因DCP1或DCP2基因的温度敏感等位基因而功能受损时，Edc3p可以刺激酵母中不稳定和稳定mRNA的脱帽。在这些情况下，在允许温度下缺失EDC3会导致协同的mRNA脱帽缺陷。当edc3Δ与lsm1Δ、dhh1Δ或pat1Δ突变结合时没有影响，这些突变似乎影响脱帽途径的早期步骤。这表明Edc3p特别影响脱帽酶本身的功能。与在脱帽中的功能作用一致，绿色荧光蛋白标记的Edc3p定位于参与mRNA脱帽的细胞质焦点，即P小体。这些结果确定Edc3p是一种参与脱帽反应的新蛋白质。
