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Analysis of translation initiation using a translation control reporter system

Nature Protocols volume 1, pages 1531–1537 (2006)Cite this article

Abstract

The study of translational control has become increasingly important, as aberrant translation has been linked to the etiology of human diseases. Nevertheless, a convenient research tool to measure and quantify cellular translational activity has not been developed to date. Here we present a translation control reporter system (TCRS) for straightforward and accurate analysis of cellular translational activity. Our method relies on the expression of two unique reporter peptides from a single messenger RNA transcript. Using TCRS-expressing cell lines, changes in initiation of translation have been detected in response to translationally active drugs. Accordingly, TCRS may promote the discovery of novel agents that modulate translation. TCRS may also be used in the identification of signal transduction pathways that impinge on translation control. Furthermore, the modular design allows the exchange of regulatory cassettes for the examination of other putative cis-regulatory mRNA elements. The time required for the procedure depends on whether transient TCRS expression is used or stable TCRS-expressing cell lines have to be produced and will range from 5 to 14 d, respectively.

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Figure 1: Design and function of the TCRS.
Figure 2: Nucleotide sequences of the TCRS.
Figure 3: Measurement of rapamycin-induced attenuation of translation by TCRS in HEK293A cells.

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Acknowledgements

This work was supported by a Max Delbrück Centre grant to V.W. and a Deutsche Forschungsgemeinschaft grant to A.L. and C.F.C. (LE 770/3-2).

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Authors and Affiliations

  1. Max Delbrück Centre for Molecular Medicine, Robert Roessle Str. 10, Berlin, D-13092, Germany

    Volker Wiesenthal & Achim Leutz

  2. Leibniz Institute for Age Research-Fritz Lipmann Institute, Beutenbergstr. 11, Jena, D-07745, Germany

    Cornelis F Calkhoven

Authors
  1. Volker Wiesenthal
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  2. Achim Leutz
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  3. Cornelis F Calkhoven
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Corresponding author

Correspondence to Cornelis F Calkhoven.

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Competing interests

The TCRS system is subject to the patent applications DE 10104952, EP 1360331, US 2004/0101888 and JP 2004-522443, assigned to the Max Delbrück Centre for Molecular Medicine (MDC) in Berlin, Germany.

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Wiesenthal, V., Leutz, A. & Calkhoven, C. Analysis of translation initiation using a translation control reporter system. Nat Protoc 1, 1531–1537 (2006). https://doi.org/10.1038/nprot.2006.274

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