Jagannathan Sujatha, Nwosu Christine, Nicchitta Christopher V
Department of Cell Biology, Duke University Medical Center, Durham, NC, USA.
Methods Mol Biol. 2011;714:301-21. doi: 10.1007/978-1-61779-005-8_19.
The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon.The partitioning of mRNAs to the ER is generally thought to be achieved by the signal recognition particle (SRP) pathway. In this pathway, mRNA localization to the ER is determined by the translation product - translation yields an N-terminal signal sequence or a topogenic signal that is recognized by the SRP and the resulting mRNA-ribosome-SRP complex is then recruited to the ER membrane. Recent studies have demonstrated that mRNAs can be localized to the ER via a signal sequence and/or translation-independent pathway(s) and that discrete sets of cytosolic protein-encoding mRNAs are enriched on the ER membrane, though they lack an encoded signal sequence. These key findings reopen investigations into the mechanism(s) that govern mRNA localization to the ER. In this contribution, we describe two independent methods that can be utilized to study this important and poorly understood aspect of eukaryotic cell biology. These methods comprise two independent means of fractionating tissue culture cells to yield free/cytosolic polyribosomes and ER membrane-bound polyribosomes. Detailed methods for the fractionation and characterization of the two polyribosome pools are provided.
分泌性和膜蛋白编码mRNA在内质网（ER）中的分配，以及它们在与ER相关的核糖体上的翻译，决定了细胞进入分泌/胞吐途径的机会。由于编码分泌性和膜蛋白的mRNA约占转录组的30%，mRNA在内质网中的定位是一种极其突出、普遍存在但却了解甚少的RNA定位现象。mRNA在内质网中的分配通常被认为是通过信号识别颗粒（SRP）途径实现的。在该途径中，mRNA在内质网中的定位由翻译产物决定——翻译产生一个N端信号序列或一个拓扑信号，该信号被SRP识别，然后产生的mRNA-核糖体-SRP复合物被招募到内质网膜上。最近的研究表明，mRNA可以通过信号序列和/或翻译非依赖途径定位于内质网，并且尽管缺乏编码的信号序列，但内质网膜上富集了一组离散的胞质蛋白编码mRNA。这些关键发现重新开启了对控制mRNA在内质网中定位机制的研究。在本论文中，我们描述了两种可用于研究真核细胞生物学这一重要但了解甚少方面的独立方法。这些方法包括两种独立的方法来分离组织培养细胞，以产生游离/胞质多核糖体和内质网膜结合的多核糖体。本文提供了两种多核糖体库的分离和表征的详细方法。
