Huch Susanne, Gommlich Jessie, Muppavarapu Mridula, Beckham Carla, Nissan Tracy
Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden.
Department of Urology, University of Rochester, Rochester, NY 14642, USA.
Sci Rep. 2016 May 5;6:25477. doi: 10.1038/srep25477.
Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.
最近有证据表明，mRNA的降解发生在正在翻译的核糖体上，或者发生在称为P小体的RNA颗粒内，P小体是一种聚集体，其核心成分是mRNA衰变蛋白和RNA。在本研究中，我们使用亚细胞分级分离法检测了mRNA脱帽蛋白Dcp1、Dcp2和Dhh1。我们发现，脱帽因子在蔗糖梯度的多核糖体组分中共沉降，并且在应激、翻译抑制或P小体形成抑制的情况下其行为不会改变。重要的是，它们在多核糖体组分中的定位与RNA无关，这表明这些因子可能组成性地定位于多核糖体。相反，加入去污剂后，脱帽蛋白的多核糖体和多核糖体后沉降被消除，去污剂将这些因子转移到非翻译的核糖核蛋白组分，这与膜结合一致。使用膜浮选分析，我们在与膜相关蛋白的浮力密度下的低密度组分中观察到了mRNA脱帽因子。这些观察结果提供了进一步的证据，表明mRNA脱帽因子与亚细胞膜相互作用，并且我们提出了一个模型，其中mRNA脱帽因子与膜相互作用以促进mRNA降解的调节。
