Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis.
10 affiliations
- 1RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld University, 33615, Bielefeld, Germany.
- 2Section for Computational Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
- 3Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.
- 4Friedrich-Löffler-Institut (FLI), Südufer 10, 17493, Greifswald, Germany.
- 5Institute of Molecular Plant Biology, Department of Biotechnology and Food Science, University of Natural Resources and Life Sciences (BOKU), Vienna, 1190, Austria.
- 6ISA - Sophia Agrobiotech Institute, Sophia Antipolis, 06903, France.
- 7Université Côte d'Azur, Nice, France.
- 8GIMM - Gulbenkian Institute for Molecular Medicine, Lisbon, Portugal.
- 9RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld University, 33615, Bielefeld, Germany. [email protected].
- 10Plant Biotechnology, Faculty of Biology, Bielefeld University, 33615, Bielefeld, Germany. [email protected].
Abstract
Background
MicroRNAs (miRNAs) play key roles in modulating gene expression. Upon transcription, primary miRNA transcripts (pri-miRNAs or MIRNAs) fold into stem-loop structures. Endonucleolytic cleavage releases a miRNA/miRNA* duplex from the stem, which is subsequently matured. In higher plants, the pri-miRNA hairpins vary widely in length and structure. Pri-miRNA processing is extensively regulated by RNA-binding proteins (RBPs) and the range of underlying mechanisms continues to expand.Results
Here, we performed an unbiased screen to identify Arabidopisis thaliana candidate RBPs that interact with pri-miRNAs, using pri-miRNA159a and pri-miRNA398b as paradigms. Nucleoplasmic proteins were significantly enriched in pulldowns with bead-immobilized in vitro transcripts of the stem-loop regions of these pri-miRNAs compared with pulldowns using empty beads. The enriched proteins included several serine/arginine-rich (SR) splicing factors, notably RS31 and SR34a, extending previous findings that RS40 and RS41 bind pri-miRNAs and participate in miRNA biogenesis. Mutant analysis indicated that selected candidate interactors affect pri-miRNA and/or mature miRNA accumulation. To validate candidate interactions with pri-miRNAs in vivo, we performed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). Whereas our conventional plant iCLIP procedure detected only a few crosslink sites on pri-miRNAs, likely because of the low abundance of these transcripts, our improved plant iCLIP2 protocol enabled the identification of binding sites on numerous pri-miRNAs. RS31 and SR34a bound overlapping sets of pri-miRNAs and contacted both stem-loop regions and flanking regions upstream and downstream of the stem-loop. Binding-site mapping resolved overlapping and distinct contact regions and highlighted the importance of complete pri-miRNA annotations for interpreting interactions beyond annotated transcript boundaries.Conclusion
In vitro pulldowns of nucleoplasmic proteins identified candidate RBPs associated with pri-miRNA stem-loops, and the improved plant iCLIP2 protocol revealed in vivo binding of RS31 and SR34a to a subset of pri-miRNAs. Together, these findings expand the repertoire of candidate RBPs implicated in plant miRNA biogenesis and support a role for SR proteins as candidate regulators of miRNA abundance.
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BibTeX
@article{winkel2026identification,
title = {{Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis.}},
author = {Andreas R Winkel and Valentin W Bitterer and Martin Lewinski and Marlene Reichel and Janina Lüders and Marion Scheibe and Maria Kalyna and Tom Laloum and Paula Duque and Dorothee Staiger and Falk Butter and Tino Köster},
journal = {BMC plant biology},
year = {2026},
month = nov,
doi = {10.1186/s12870-026-10297-z},
url = {https://europepmc.org/article/MED/42972225},
note = {CC BY},
}
RIS
TY - JOUR
TI - Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis.
AU - Andreas R Winkel
AU - Valentin W Bitterer
AU - Martin Lewinski
AU - Marlene Reichel
AU - Janina Lüders
AU - Marion Scheibe
AU - Maria Kalyna
AU - Tom Laloum
AU - Paula Duque
AU - Dorothee Staiger
AU - Falk Butter
AU - Tino Köster
PY - 2026
DA - 2026/11/02
JO - BMC plant biology
DO - 10.1186/s12870-026-10297-z
UR - https://europepmc.org/article/MED/42972225
AB - Background MicroRNAs (miRNAs) play key roles in modulating gene expression. Upon transcription, primary miRNA transcripts (pri-miRNAs or MIRNAs) fold into stem-loop structures. Endonucleolytic cleavage releases a miRNA/miRNA* duplex from the stem, which is subsequently matured. In higher plants, the pri-miRNA hairpins vary widely in length and structure. Pri-miRNA processing is extensively regulated by RNA-binding proteins (RBPs) and the range of underlying mechanisms continues to expand.Results Here, we performed an unbiased screen to identify Arabidopisis thaliana candidate RBPs that interact with pri-miRNAs, using pri-miRNA159a and pri-miRNA398b as paradigms. Nucleoplasmic proteins were significantly enriched in pulldowns with bead-immobilized in vitro transcripts of the stem-loop regions of these pri-miRNAs compared with pulldowns using empty beads. The enriched proteins included several serine/arginine-rich (SR) splicing factors, notably RS31 and SR34a, extending previous findings that RS40 and RS41 bind pri-miRNAs and participate in miRNA biogenesis. Mutant analysis indicated that selected candidate interactors affect pri-miRNA and/or mature miRNA accumulation. To validate candidate interactions with pri-miRNAs in vivo, we performed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). Whereas our conventional plant iCLIP procedure detected only a few crosslink sites on pri-miRNAs, likely because of the low abundance of these transcripts, our improved plant iCLIP2 protocol enabled the identification of binding sites on numerous pri-miRNAs. RS31 and SR34a bound overlapping sets of pri-miRNAs and contacted both stem-loop regions and flanking regions upstream and downstream of the stem-loop. Binding-site mapping resolved overlapping and distinct contact regions and highlighted the importance of complete pri-miRNA annotations for interpreting interactions beyond annotated transcript boundaries.Conclusion In vitro pulldowns of nucleoplasmic proteins identified candidate RBPs associated with pri-miRNA stem-loops, and the improved plant iCLIP2 protocol revealed in vivo binding of RS31 and SR34a to a subset of pri-miRNAs. Together, these findings expand the repertoire of candidate RBPs implicated in plant miRNA biogenesis and support a role for SR proteins as candidate regulators of miRNA abundance.
SN - 1471-2229
ER -