Single mRNA interactome capture in Arabidopsis - RAPping rhythms
Final Report Abstract
The project aimed at determining the inventory of proteins interacting with a single mRNA in plants, using the highly expressed AtGRP7 transcript as a paradigm. We systematically optimized the RNA interactome capture protocol developed to pull down the mRNA complement using oligo (dT) beads, followed by MS of the eluted proteins. Several aspects have proven critical for the success of the approach: The design of AOs is key for efficient recovery of specific mRNAs. In particular, the use of LNA antisense oligonucleotides was superior to regular oligonucleotides. Furthermore, an optimal UV dosage is important. We tested different combinations of affinity capture steps, using a specific AO hybridizing to the 5´UTR and LNA 2.T oligo nucleotides. Finally, we performed two rounds of capture using the 5´UTR oligo. Despite the use of 100 g tissue as starting material we only identified a limited number of proteins, indicating that massive upscaling and more sensitive MS will be required. To validate the identified proteins by independent means, we performed an in vitro pulldown using immobilized in vitro transcripts comprising the AtGRP7 3´UTR and 5´UTR. Amongst the proteins common to in vivo and in vitro approaches were the ALBA protein family. The AtGRP7 transcript was independently verified as target of ALBA by iCLIP, with strong binding sites in the AtGRP7 5’UTR and especially in the 3’UTR. Subtle differences in AtGRP7 expression in alba mutants compared to Col-0 suggested that ALBA proteins may influence AtGRP7. In the future, additional candidate AtGRP7 binding proteins can be validated using the same workflow.
Publications
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CLIP and RNA interactome studies to unravel genomewide RNA-protein interactions in vivo in Arabidopsis thaliana. Methods 178, 63-71
Köster T., Reichel M. & Staiger D.
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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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Mapping protein–RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2). Nature Protocols, 19(4), 1183-1234.
Lewinski, Martin; Brüggemann, Mirko; Köster, Tino; Reichel, Marlene; Bergelt, Thorsten; Meyer, Katja; König, Julian; Zarnack, Kathi & Staiger, Dorothee
