Abstract
It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5′-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.
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Acknowledgments
We would like to thank Avery Zierk for technical support. This work was supported in part by National Institutes of Health Grants [1R01CA172567-01A1 to T.H.] and in part supported by pilot research funding from an American Cancer Society Institutional Research Grant awarded to the Hollings Cancer Center, Medical University of South Carolina [IRG-97-219-11 to T.H.]. We thank the Verein zur Förderung krebskranker und körperbehinderter Kinder Ostbayern e.V. (VKKK) for their support.
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Sommer, G., Sendlmeier, C., Heise, T. (2020). Salt-Dependent Modulation of the RNA Chaperone Activity of RNA-Binding Protein La. In: Heise, T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_7
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DOI: https://doi.org/10.1007/978-1-0716-0231-7_7
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