Gandin Valentina, Sikström Kristina, Alain Tommy, Morita Masahiro, McLaughlan Shannon, Larsson Ola, Topisirovic Ivan
Lady Davis Institute and Department of Oncology, McGill University.
Department of Oncology-Pathology, Karolinska Institutet.
J Vis Exp. 2014 May 17(87):51455. doi: 10.3791/51455.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed "anota" algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
mRNA翻译在基因表达调控中起着核心作用，是哺乳动物细胞中最耗能的过程。因此，mRNA翻译失调被认为在包括癌症在内的多种病理状态中起主要作用。核糖体还含有分子伴侣，这些分子伴侣促进新生多肽的折叠，从而调节新合成多肽的功能和稳定性。此外，新出现的数据表明，核糖体是一系列信号分子的平台，这些信号分子与新合成的多肽从核糖体中出现时的各种翻译后修饰以及翻译机制的组成部分有关。本文描述了一种使用蔗糖密度梯度离心进行核糖体分级分离的成熟方法。结合内部开发的“anota”算法，该方法可以在全基因组范围内直接测定单个mRNA的差异翻译。此外，这个通用方案可用于各种生化研究，旨在剖析核糖体相关蛋白复合物的功能，包括那些在新合成多肽的折叠和降解中起核心作用的复合物。
