Abstract
Basic research in Drosophila melanogaster has benefited from a plethora of powerful genetics tools. Detailed biochemical analysis, however, has often been difficult due to the lack of in vitro systems that faithfully recapitulate the observations made in vivo. In the field of posttranscriptional regulation, the recent establishment of robust in vitro systems from embryo and ovary material has fueled the mechanistic understanding of a variety of processes. Here we describe protocols to obtain and use extracts from Drosophila embryos that are competent for cytoplasmic polyadenylation and translation of exogenously added transcripts.
An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-62703-971-0_29
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-62703-971-0_29
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
Gebauer F, Corona DF, Preiss T et al (1999) Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5′ and 3′ UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J 18:6146–6154
Castagnetti S, Hentze MW, Ephrussi A et al (2000) Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 127:1063–1068
Lie YS, Macdonald PM (2000) In vitro translation extracts prepared from Drosophila ovaries and embryos. Biochem Biophys Res Commun 270:473–481
Andrews S, Snowflack DR, Clark IE et al (2011) Multiple mechanisms collaborate to repress nanos translation in the Drosophila ovary and embryo. RNA 17:967–977
Thermann R, Hentze MW (2007) Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447:875–878
Jeske M, Meyer S, Temme C et al (2006) Rapid ATP-dependent deadenylation of nanos mRNA in a cell-free system from Drosophila embryos. J Biol Chem 281:25124–25133
Hernandez G, Vazquez-Pianzola P, Sierra JM et al (2004) Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos. RNA 10:1783–1797
Tuschl T, Zamore PD, Lehmann R et al (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191–3197
Gebauer F, Hentze MW (2007) Studying translational control in Drosophila cell-free systems. Methods Enzymol 429:23–33
Jeske M, Wahle E (2008) Cell-free deadenylation assays with Drosophila embryo extracts. Methods Enzymol 448:107–118
Haley B, Tang G, Zamore PD (2003) In vitro analysis of RNA interference in Drosophila melanogaster. Methods 30:330–336
Lipardi C, Baek HJ, Wei Q et al (2005) Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells. Methods Enzymol 392:351–371
Villalba A, Coll O, Gebauer F (2011) Cytoplasmic polyadenylation and translational control. Curr Opin Genet Dev 21:452–457
Fernandez-Miranda G, Mendez R (2012) The CPEB-family of proteins, translational control in senescence and cancer. Age Res Rev 11(4): 460–472. doi:10.1016/j.arr.2012.03.004
Coll O, Villalba A, Bussotti G et al (2010) A novel, noncanonical mechanism of cytoplasmic polyadenylation operates in Drosophila embryogenesis. Genes Dev 24:129–134
Salles FJ, Lieberfarb ME, Wreden C et al (1994) Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266: 1996–1999
Salles FJ, Strickland S (1995) Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR Meth Appl 4:317–321
Charlesworth A, Cox LL, MacNicol AM (2004) Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes. J Biol Chem 279: 17650–17659
Acknowledgments
A. V. is supported by an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. Work in our lab is supported by grants BFU2009-08243 and Consolider CSD2009-00080 from the same ministry.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Coll, O., Villalba, A., Gebauer, F. (2014). Cytoplasmic Polyadenylation Assays. In: Rorbach, J., Bobrowicz, A. (eds) Polyadenylation. Methods in Molecular Biology, vol 1125. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-971-0_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-971-0_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-970-3
Online ISBN: 978-1-62703-971-0
eBook Packages: Springer Protocols