Abstract
Trypanosomatids rely primarily on posttranscriptional mechanisms for the control of gene expression, with regulation of RNA processing, localization, degradation, and translation by RNA-binding proteins (RBPs). To determine the mechanisms by which RBPs control gene expression in trypanosomatids, transcriptome-wide identification of mRNA targets and mapping of the RNA-binding site is required. Here we present our most current RIP-Seq (RNA immunoprecipitation followed by high-throughput sequencing) protocol that we generally apply to elucidate RNA/protein interactions in Trypanosoma brucei. The technique provides valuable information about the workings of messenger ribonucleoprotein (mRNP) networks and trypanosome gene expression mechanisms.
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References
Tenenbaum SA, Carson CC, Lager PJ et al (2000) Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A 97:14085–14090
Licatalosi DD, Mele A, Fak JJ et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469
Hafner M, Landthaler M, Burger L et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Konig J, Zarnack K, Rot G et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915
Mili S, Steitz JA (2004) Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10:1692–1694
Droll D, Minia I, Fadda A et al (2013) Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein. PLoS Pathog 9:e1003286
Minia I, Clayton C (2016) Regulating a post-transcriptional regulator: protein phosphorylation, degradation and translational blockage in control of the trypanosome stress-response RNA-binding protein ZC3H11. PLoS Pathog 12:e1005514
Minia I, Merce C, Terrao M et al (2016) Translation regulation and RNA granule formation after heat shock of procyclic form Trypanosoma brucei: many heat-induced mRNAs are also increased during differentiation to mammalian-infective forms. PLoS Negl Trop Dis 10:e0004982
Mugo E, Clayton C (2017) Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei. PLoS Pathog 13:e1006560
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106
Kucukural A, Ozadam H, Singh G et al (2013) ASPeak: an abundance sensitive peak detection algorithm for RIP-Seq. Bioinformatics 29:2485–2486
Li Y, Zhao DY, Greenblatt JF et al (2013) RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-Seq experiments. Nucleic Acids Res 41:e94
Bailey TL, Boden M, Buske FA et al (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208
Achar A, Saetrom P (2015) RNA motif discovery: a computational overview. Biol Direct 10:61
Aslett M, Aurrecoechea C, Berriman M et al (2010) TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38:D457–D462
Najafabadi HS, Lu Z, MacPherson C et al (2013) Global identification of conserved post-transcriptional regulatory programs in trypanosomatids. Nucleic Acids Res 41:D8591–D8600
Puig O, Caspary F, Rigaut G et al (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229
Acknowledgments
We thank Christine Clayton for her continuous support and all former and current CC lab members who have contributed to improve this and many other techniques.
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Mugo, E., Erben, E.D. (2020). Identifying Trypanosome Protein–RNA Interactions Using RIP-Seq. 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_17
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DOI: https://doi.org/10.1007/978-1-0716-0294-2_17
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