Abstract
The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient “handle” for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.
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References
Keene, J. D. (2007) RNA regulons: coordination of post-transcriptional events. Nat Rev Genet 8, 533–543.
Gerber, A. P., Herschlag, D., Brown, P. O. (2004) Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol 2, E79.
Krichevsky, A. M., Kosik, K. S. (2001) Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron 32, 683–696.
Kanai, Y., Dohmae, N., Hirokawa, N. (2004) Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43, 513–525.
Villace, P., Marion, R. M., Ortin, J. (2004) The composition of Staufen-containing RNA granules from human cells indicates their role in the regulated transport and translation of messenger RNAs. Nucleic Acids Res 32, 2411–2420.
Elvira, G., Wasiak, S., Blandford, V., Tong, X. K., Serrano, A., Fan, X., del Rayo Sanchez-Carbente, M., Servant, F., Bell, A. W., Boismenu, D., Lacaille, J. C., McPherson, P. S., DesGroseillers, L., Sossin, W. S. (2006) Characterization of an RNA granule from developing brain. Mol Cell Proteomics 5, 635–651.
Mili, S., Steitz, J. A. (2004) Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10, 1692–1694.
Jonson, L., Vikesaa, J., Krogh, A., Nielsen, L. K., Hansen, T., Borup, R., Johnsen, A. H., Christiansen, J., Nielsen, F. C. (2007) Molecular composition of IMP1 ribonucleoprotein granules. Mol Cell Proteomics 6, 798–811.
Acknowledgements
This work was supported by the Danish Natural Science and Medical Research Councils, and the Lundbeck Foundation.
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Jønson, L., Nielsen, F.C., Christiansen, J. (2011). Isolation of RNP Granules. In: Nielsen, H. (eds) RNA. Methods in Molecular Biology, vol 703. Humana Press. https://doi.org/10.1007/978-1-59745-248-9_18
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DOI: https://doi.org/10.1007/978-1-59745-248-9_18
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