Regulation of miRNA biogenesis in Arabidopsis - An RNA-affinity based approach to characterize pri-miRNA stem-loop binding proteins
Final Report Abstract
Regulation at the level of RNA-binding proteins (RBPs) and miRNAs is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long pri-miRNAs with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we established RNA-centric approaches to pull down interactors of selected pri-miRNA stem-loops in vitro. First, we exploited an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4*. To overcome technical difficulties associated with the Csy4* system, we additionally set up a powerful Streptavidin-based RNA-affinity purification strategy. Protein interactors of pri-miRNA stem-loops were identified via quantitative mass spectrometry (MS). In total, we identified hundreds of interactors with a putative regulatory function in miRNA biogenesis. To proof their physiological relevance in miRNA biogenesis, loss- and gain-of-function mutants of selected RBPs were genotyped and analyzed. Among others, we could show that members of the glycine-rich, serine-arginine (SR) rich and RNA helicase family regulate the biogenesis of specific miRNAs. To obtain genome-wide insights into the extent and molecular mechanism of RBP action, we identified direct in vivo targets and RBP binding sites of the RS family protein RS31 by plant individual nucleotide resolution crosslinking and immunoprecipitation (plant iCLIP). Overall, the project delivers new insight into molecular mechanisms underlying posttranscriptional regulation of miRNA processing by plant RBPs and helps to understand the regulatory principles of eukaryotic miRNA expression.
Publications
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“Genome-wide determination of RNA-binding protein targets in Arabidopsis“ Invited speaker at IGC symposium 2017 “Plant RNA biology”, Oieras, Portugal (2017)
Tino Köster
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Plant Ribonomics: Proteins in Search of RNA Partners. Trends in Plant Science, 23(4), 352-365.
Köster Tino & Meyer Katja
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Systems Approaches to Map In Vivo RNA–Protein Interactions in Arabidopsis thaliana. RNA Technologies, 77-95. Springer International Publishing.
Lewinski, Martin & Köster, Tino
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CLIP and RNA interactome studies to unravel genome-wide RNA-protein interactions in vivo in Arabidopsis thaliana. Methods, 178, 63-71.
Köster, Tino; Reichel, Marlene & Staiger, Dorothee
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CLIP and complementary methods. Nature Reviews Methods Primers, 1(1).
Hafner, Markus; Katsantoni, Maria; Köster, Tino; Marks, James; Mukherjee, Joyita; Staiger, Dorothee; Ule, Jernej & Zavolan, Mihaela
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“Profiling a new set of nucleoplasmic players in miRNA biogenesis” DBG hot topic meeting “It’s in your RNA”, Bielefeld, Germany (2021)
Tino Köster
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Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease. International Journal of Molecular Sciences, 23(16), 8961.
Lüders, Janina; Winkel, Andreas R.; Reichel, Marlene; Bitterer, Valentin W.; Scheibe, Marion; Widmann, Christiane; Butter, Falk & Köster, Tino
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Plant iCLIP — auf Spurensuche im Transkriptom der Pflanze. BIOspektrum, 29(2), 174-176.
Köster, Tino & Sitte, Astrid
