Characterization of microRNA-mediated cleavage events in Arabidopsis embryos

Aleksandra Plotnikova
2021 unpublished
MicroRNAs (miRNAs) are small regulatory RNAs that repress genes in plants and animals. Previously we found that miRNA-deficient Arabidopsis embryos exhibit widespread cellular differentiation and developmental timing defects (Nodine and Bartel, 2010). However, little was known about the contributions of individual miRNAs to pattern formation. To systematically characterize miRNA functions during embryogenesis, we profiled small RNAs throughout embryogenesis and identified dozens of miRNA
more » ... s that are abundant during the morphogenesis phase of embryo development. The functions of miRNAs are defined by the genes they regulate. In plants, miRNAs have nearly perfect complementarity to their targets and typically guide their endonucleolytic cleavage. We have developed a high-throughput method, nanoPARE (parallel analysis of RNA ends), to profile miRNA cleavage products genome-wide from sub-nanogram amounts of total RNA. We applied nanoPARE to early embryos and identified dozens of miRNA:target interactions operating during embryogenesis. Our nanoPARE data, together with transcriptome analysis of wild-type and miRNA-deficient embryos, enabled the identification of several miRNA families that cleave and repress distinct sets of transcription factors. Because miRNA-mediated regulation of transcription factors may be especially crucial for embryonic pattern formation, we selected these miRNAs for further characterization. We used fluorescent protein-based methods to determine the activity patterns of these miRNAs at cellular resolution during embryogenesis. In addition, we generated transgenic plants expressing miRNA-resistant versions of these target transcription factors and examined the morphology of embryos to determine whether cell-specific repression of transcription factors is required for proper embryo morphogenesis. Altogether, our results demonstrate that miRNAs define the spatiotemporal localization of transcription factors, which is determinative for establishing the body plan at the beginning of plant li [...]
doi:10.25365/thesis.65899 fatcat:2tug4yd2ifdvhmjexyxodojlpa