Summary
Regulating gene expression at the translational level controls a wide variety of biological events such as development, long-term memory, stress response, transport and storage of certain nutrients, and viral infection. Protein synthesis at steady-state level can be directly measured with Western blot or using an easy-to-detect reporter such as luciferase. However, these methods do not measure the association of mRNA with ribosomes, which is more meaningful in understanding the mechanism and dynamics of translation. This chapter describes the use of sucrose density gradients for analysis of polysome profiles. RNA or protein samples extracted from gradient fractions are commonly used for further analysis of their association with translating ribosomes. We also describe an in vitro translation system prepared from HeLa S3 cell cytoplasmic extract that shows dependency on the mRNA cap and length of the poly(A) length tail, both features of translation in vivo. This is particularly useful to study the cis- and trans-acting factors involved in translational control. Lastly, we describe a method for transfecting cells with an in vitro prepared RNA to study the impact of poly(A) length on translation. This approach is particularly useful for characterizing cis-acting elements that work in conjunction with poly(A) in regulating translation.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O., and Herschlag, D (2003) Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 100, 3889–3894.
Raught, B., Gingras, A. C., and Sonenberg, N (2000) Regulation of ribosomal recruitment in eukaryotes, in Translational Control of Gene ExpressionSonenberg, N., Hershey, J. W. B., Mathews, M. B., and Ab, G., eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 245–293.
Mangus, D. A., Evans, M. C., and Jacobson, A (2003) Poly(A) binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol. 4, 223.
Sachs, A. B (2000) Physical and functional interactions between the mRNA cap structure and the poly(A) tail, in Translational Control of Gene ExpressionSonenberg, N., Hershey, J. W. B., Mathews, M. B., and Ab, G., eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 447–465.
Wahle, E. and Keller, W (1996) The biochemistry of polyadenylation. Trends Biochem. Sci. 21, 247–250.
Tharun, S. and Parker, R (1997) Mechanisms of mRNA turnover in eukaryotic cells, in mRNA Metabolism and Post-transcriptional Gene Regulation (Harford, J. and Morris, D. R., eds). Wiley, New York, pp. 181–200.
Tharun, S. and Parker, R (2001) Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Mol. Cell 8, 1075–1083.
Sheth, U. and Parker, R (2003) Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808.
Sachs, A. B (1987) A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7, 3268–3276.
Preiss, T., Muckenthaler, M., and Hentze, M. W. (1998) Poly(A)-tail-promoted translation in yeast: implications for translational. RNA 4, 1321–1331.
Bergamini, G., Preiss, T., and Hentze, M. W (2000) Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA 6, 1781–1790.
Peng, J. and Schoenberg, D. R (2005) mRNA with a <20 nt poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA. RNA 11, 1131–1140.
Gallie, D. R (1991) The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 5, 2108–2116.
Grudzien, E., Kalek, M., Jemielity, J., Darzynkiewicz, and Rhoads, R. E (2006) Differential inhibition of mRNA degradation pathways by novel cap analogs J. Biol. Chem. 281, 1857–1867.
Kahvejian, A., Svitkin, Y. V., Sukarieh, R., M’Boutchou, M. N., and Sonenberg, N (2005) Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes Dev. 19, 104–113.
Acknowledgments
This work was supported by PHS grant R01 GM38277 and GM55407 to D.R.S. E.L.M. was supported in part by PHS grant T32 CA09338, and support for core facilities was provided by PHS center grant P30 CA16058 from the National Cancer Institute to The Ohio State University Comprehensive Cancer Center.
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
Peng, J., Murray, E.L., Schoenberg, D.R. (2008). In Vivo and In Vitro Analysis of Poly(A) Length Effects on mRNA Translation. 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_15
Download citation
DOI: https://doi.org/10.1007/978-1-59745-033-1_15
Publisher Name: Humana Press
Print ISBN: 978-1-58829-783-9
Online ISBN: 978-1-59745-033-1
eBook Packages: Springer Protocols