Analyzing mRNA Localization to the Endoplasmic Reticulum via Cell Fractionation

摘要: The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), their translation on ER-associated ribosomes, governs access secretory/exocytic pathways cell. As encoding proteins comprise approximately 30% transcriptome, localization ER represents an extraordinarily prominent, ubiquitous, yet poorly understood RNA phenomenon.The is generally thought be achieved by signal recognition particle (SRP) pathway. In this pathway, mRNA determined product - yields N-terminal sequence or a topogenic that recognized SRP resulting mRNA-ribosome-SRP complex then recruited membrane. Recent studies have demonstrated can localized via and/or translation-independent pathway(s) discrete sets cytosolic are enriched membrane, though they lack encoded sequence. These key findings reopen investigations into mechanism(s) govern ER. contribution, we describe two independent methods utilized study important aspect eukaryotic cell biology. means fractionating tissue culture cells yield free/cytosolic polyribosomes membrane-bound polyribosomes. Detailed for fractionation characterization polyribosome pools provided.