Regulation of miRNA biogenesis in Arabidopsis - An RNA-affinity based approach to characterize pri-miRNA stem-loop binding proteins

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Regulation at the RNA level by RNA-binding proteins (RBPs) and small regulatory RNAs is key to coordinating eukaryotic gene expression. Among small RNAs are microRNAs (miRNAs) that are ~21nt-long single-stranded RNAs and regulate the expression of cognate target mRNAs by cleavage or inhibition of translation. The importance of miRNAs is highlighted by their regulatory functions in development, differentiation and environmental responses. MiRNAs are generated from double-stranded primary (pri)-miRNAs with internal stem-loop structures that are converted to short-lived stem-loop precursor (pre)-miRNAs and further processed to mature miRNAs. In contrast to their mammalian counterparts, stem-loops of plant miRNA precursors are diverse and highly structured which constitutes a basis for extensive regulation of miRNA biogenesis through direct binding of RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown. Our goal is a comprehensive identification of proteins that bind to the stem-loops of pri-miRNAs and affect miRNA biogenesis in Arabidopsis thaliana. We will exploit an RNA-centric approach to pull down proteins directly binding to the stem-loops of selected miRNA precursors in vitro. Therefore, in vitro transcripts of selected pri-miRNA stem-loops will be tagged with the short recognition sequence of the CRISPR endoribonuclease Csy4. An enzymatically inactive variant, Csy4*, will be expressed in E.coli and affinity-purified. After biotinylation, Csy4* will be immobilized on streptavidin beads and the Csy4* beads will be loaded with the pri-miRNA stem-loops tagged with the Csy4 recognition sequence. The Csy4*-RNA beads will then be incubated with protein extracts prepared from Arabidopsis nuclei to allow binding of RBPs to the pri-miRNA stem-loops. Upon reactivation of the Csy4* nuclease activity by imidazole the Csy4* recognition sequence is cleaved off the pri-miRNA stem-loops and the RNA-protein complexes are released. The method for this affinity enrichment will be optimized based on the specific enrichment of the known trans-acting regulators of miRNA biogenesis and general processing factors binding to pri-miR398b. Subsequently, the procedure will be scaled up and co-precipitating proteins will be identified via mass spectrometry. Proteins which are significantly enriched with tagged pri-miRNAs loaded onto the Cys4* beads but not by beads carrying only the tag are candidates regulating pri-miRNA processing. Their physiological relevance in miRNA biogenesis will be analyzed in loss-of-function mutants. To obtain genome-wide insights into the extent and molecular mechanism of RBP action, direct in vivo targets and RBP binding sites will be identified.Overall, the project will deliver new insight into molecular mechanisms underlying posttranscriptional regulation of miRNA processing by plant RBPs and will ultimately advance our understanding of regulatory principles of eukaryotic miRNA expression.