Abstract
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.
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Acknowledgements
The authors thank Angela Jockheck-Clark for critical reading of the manuscript. We thank Tianli Zheng and other present and past members of the Nicchitta laboratory for their contribution to the development of the techniques described in this article. We also wish to thank Mayya Shveygert and the Gromeier lab for providing GAPDH and H3 antibodies. This work was supported by NIH grant GM-077382 to CVN.
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Jagannathan, S., Nwosu, C., Nicchitta, C.V. (2011). Analyzing mRNA Localization to the Endoplasmic Reticulum via Cell Fractionation. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_19
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DOI: https://doi.org/10.1007/978-1-61779-005-8_19
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