Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease

Abstract

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (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 primary-microRNAs (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 exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.