Summary
Cytoplasmic extracts have proven to be a versatile system for assaying the mechanisms and interactions of RNA metabolism. Using Aedes albopictus (C6/36) cells adapted to suspension culture, we have been able to faithfully reproduce and manipulate all aspects of mRNA decay in vitro. Described in this chapter are the processes for both producing an active cytoplasmic extract and the subsequent applications of the extract with respect to mRNA decay. The following protocol for the production of cytoplasmic extracts from C6/36 cells can be altered to encompass a wide variety of cell types, including mammalian cell lines. In addition, a method for designing and implementing an in vitro transcription template to produce specific products are described in detail. Applications of the in vitro transcripts, specifically the deadenylation and exosome assays by which the decay of reporter transcripts is observed, are also examined in detail.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cheadle, C., Fan, J, Cho-Chung, Y.S., Werner, T., Ray, J., Do, L., Gorospe, M., and Becker, K.G. (2005) Stability regulation of mRNA and the control of gene expression. Ann N Y Acad Sci 1058:196–204.
Amrani, N., Sachs, M.S., and Jacobson, A. Early nonsense: mRNA decay solves a translational problem. (2006) Nat Rev Mol Cell Biol 7:415–425.
van Hoof, A., Frischmeyer, P.A., Dietz, H.C., and Parker, R. (2002) Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science 295:2262–2264.
Doma, M.K. and Parker, R. (2006) Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440:561–564.
Anderson, P. and Kedersha, N. (2006) RNA granules. J Cell Biol 172:803–808.
Ford, L.P., Watson, J., Keene, J.D., and Wilusz, J. (1999) ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system. Genes Dev 13:188–201.
Wilusz, C.J., Gao, M., Wilusz, J., and Peltz, S.W. (2001) Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts. RNA 7:1416–1424.
Milone, J., Wilusz, J., and Bellofatto, V. (2004) Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p. RNA 10:448–457.
Opyrchal, M., Anderson, J.R., Sokoloski, K.J., Wilusz, C.J., and Wilusz, J. (2005) A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines. Insect Biochem Mol Biol 35:1321–1334.
Conti, E. and Izaurralde, E. (2005) Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species. Curr Opin Cell Biol 17: 316–325.
Acknowledgments
We thank all members, past and present, of the Wilusz laboratory who have contributed to the development of the in vitro mRNA decay system in a multitude of cell lines and organisms. This work was supported by NIH grants GM072481 and AI 063434 to J. W.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sokoloski, K., Anderson, J.R., Wilusz, J. (2008). Development of an In Vitro mRNA Decay System in Insect Cells. In: Wilusz, J. (eds) Post-Transcriptional Gene Regulation. Methods In Molecular Biology™, vol 419. Humana Press. https://doi.org/10.1007/978-1-59745-033-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-59745-033-1_19
Publisher Name: Humana Press
Print ISBN: 978-1-58829-783-9
Online ISBN: 978-1-59745-033-1
eBook Packages: Springer Protocols