MicroRNAs (miRNAs) are tiny RNA molecules that regulate gene expression by controlling messenger RNAs (mRNAs) and are critical for various biological processes, including development, stress responses, and epigenetic regulation. In plants, the enzyme DICER-LIKE 1 (DCL1) processes miRNA precursors into mature miRNAs, and mutations in DCL1 can lead to developmental issues like delayed flowering and abnormal leaves, making its precision essential for plant growth. A research team led by Prof. NGUYEN Tuan Anh from the Division of Life Science at HKUST has recently made significant strides in understanding miRNA biogenesis by developing a groundbreaking massively parallel dicing assay to investigate human DICER, which functions similarly to plant DCL1.
Innovative Approaches Unveils the GHR Motif in Plant miRNA Processing
This innovative assay incorporates thousands of RNA substrates and uses deep sequencing and computational analysis to identify RNA elements that control DICER cleavage accuracy and efficiency. Building on this expertise, Prof. Nguyen's team applied their dicing assay to explore how DCL1 determines its cleavage sites in miRNA precursors. In collaboration with Dr. CHEN Xuemei's group from Peking University, their study revealed key RNA structural and sequence elements that determine DCL1's precision and efficiency, focusing on the discovery and characterization of cis-RNA elements that guide DCL1's cleavage activity.
This pioneering study identified the GHR motif as a critical determinant of DCL1's cleavage specificity (Fig. 1a), utilizing a randomized RNA library of over 46,000 sequences and an innovative dicing assay to map DCL1's preferences. The GHR motif operates independently of DCL1's dsRBD and helicase regions, influencing cleavage through the RIIIDa domain, which reveals a new mechanism of DCL1's interaction with RNA substrates. Additionally, the GHR motif is conserved among various plant species, underscoring its evolutionary importance in miRNA processing. Notably, the core component of the GHR motif, the C-G base pair, is also found in the cleavage sites of two animal RNase III enzymes: the double YCR motif for Dicer2,3 and the DRES motif for Drosha4 (Fig. 1b).
The study also presents an alternative mechanism in plants for producing non-canonical 22-nt miRNAs, facilitated by the GHR motif. This discovery adds a significant layer of complexity to miRNA length variation and highlights its regulatory implications for gene expression across diverse plant systems.
GHR Motif: Advancing Our Understanding of Gene Regulation and Plant Development
The insights gained from the GHR motif allow for precise engineering of miRNA pathways, potentially improving traits such as flowering time, stress tolerance, and crop yields. The introduction of a new pathway for producing 22-nt miRNAs, which are essential for triggering secondary RNA interference and regulating complex gene networks, opens new avenues for understanding the broader functions of gene regulation by miRNAs. Furthermore, the innovative massively parallel dicing assay and GHR motif analysis can be applied to studies of other DICER-like enzymes, enhancing our understanding of RNA-based gene regulation. As Prof. Nguyen stated, "This research significantly advances our understanding of miRNA biogenesis and highlights the critical role of RNA elements in gene regulation and plant development. The discoveries made in this study provide valuable insights into the evolutionary conservation of miRNA processing mechanisms and contribute to the development of innovative approaches for crop improvement."
The research findings have been published in Nature Plants.
