Abstract
RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.
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
Similar content being viewed by others
References
Zhao BS, Roundtree IA, He C (2017) Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol 18:31–42
Clayton C (2013) The regulation of trypanosome gene expression by RNA-binding proteins. PLoS Pathog 9:e1003680
Glisovic T, Bachorik JL, Yong J, Dreyfuss G (2008) RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 582:1977–1986
Lee FCY, Ule J (2018) Advances in CLIP technologies for studies of protein-RNA interactions. Mol Cell 69:354–369
Ule J, Jensen KB, Ruggiu M et al (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215
König J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915
Dixit S, Müller-McNicoll M, David V, Zarnack K, Ule J, Hashimi H, Lukeš J (2017) Differential binding of mitochondrial transcripts by MRB8170 and MRB4160 regulates distinct editing fates of mitochondrial mRNA in trypanosomes. mBio 8:e02288-16
Dixit S, Lukeš J (2018) Combinatorial interplay of RNA-binding proteins tunes levels of mitochondrial mRNA in trypanosomes. RNA 24:1594–1606
Huppertz I, Attig J, D’Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Konig J, Ule J (2014) iCLIP: protein–RNA interactions at nucleotide resolution. Methods 65:274–287
Acknowledgments
Support from the Czech Grant Agency (18-15962S), ERC CZ (LL1601) and the ERD Funds, project OPVVV (16_019/0000759) (all to J.L.) is acknowledged.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dixit, S., Alfonzo, J.D., Lukeš, J. (2020). RNA-Binding Proteins and Their Targets in Trypanosoma brucei: Single Nucleotide Resolution Using iCLIP and iCLAP. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0294-2_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0294-2_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0293-5
Online ISBN: 978-1-0716-0294-2
eBook Packages: Springer Protocols