Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis

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.