Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.
Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.
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DOI：
10.1007/978-1-61779-005-8_19
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发表时间：
2011
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影响因子：
--
通讯作者：
Nicchitta, Christopher V
中科院分区：
文献类型：
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作者：
Jagannathan, Sujatha;Nwosu, Christine;Nicchitta, Christopher V
分泌和膜蛋白编码mRNA分配到内质网（ER），以及它们在ER相关核糖体上的翻译，控制着进入细胞的分泌/胞外途径。由于编码分泌蛋白和膜蛋白的mRNA占转录组的约30%，mRNA定位于ER代表了一种非常突出、普遍存在但知之甚少的RNA定位现象。mRNA分配到ER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定位于ER由翻译产物决定-翻译产生被SRP识别的N-末端信号序列或拓扑信号，然后将所得的mRNA-核糖体-SRP复合物募集至ER膜。最近的研究表明，mRNA可以通过信号序列和/或非依赖性途径定位于ER，并且细胞溶质蛋白编码mRNA的离散集合在ER膜上富集，尽管它们缺乏编码的信号序列。这些关键发现重新开启了对mRNA定位于ER的机制的研究。在这方面的贡献，我们描述了两个独立的方法，可用于研究这一重要的和了解甚少的真核细胞生物学方面。这些方法包括两种分离组织培养细胞以产生游离/胞质多核糖体和ER膜结合多核糖体的独立方法。提供了用于两个多核糖体池的分级分离和表征的详细方法。
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 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.