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Determinants of selectivity in the dicing mechanism

ABSTRACT: Determinants of selectivity in the dicing mechanism

PROVIDER: PRJNA1060648 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27407651_1.fastq.gz	Fastqsanger.gz
	SRR27407651_2.fastq.gz	Fastqsanger.gz
	SRR27407652_1.fastq.gz	Fastqsanger.gz
	SRR27407652_2.fastq.gz	Fastqsanger.gz
	SRR27407653_1.fastq.gz	Fastqsanger.gz

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Determinants of selectivity in the dicing mechanism

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Determinants of selectivity in the dicing mechanism

Project description:The RNase III enzyme, DICER, is instrumental in the production of small RNAs, including miRNAs and siRNAs, by cleaving their precursors, such as pre-miRNAs, shRNAs, and long duplex RNAs. Utilizing High-throughput (HT) cleavage assays, our study delves into the cleavage activity of DICER. We challenge the widely accepted 2-nt loop counting rule in the previous study, revealing a divergent mechanism, the bipartite base pairing rule. This rule directs DICER's cleavage sites via the RNase III domain. Moreover, we demystify the recognition mechanism of the previously identified YCR motif. Building on this understanding, a secondary YCR motif that also influences DICER's cleavage sites has been discovered. We also address a long-debated issue concerning DICER's cleavage sites on long stem RNAs, such as pre-siRNAs or long shRNAs/pre-miRNAs. Our study shows that the dsRBD plays a crucial role in determining the cleavage sites of DICER in long-stem RNAs. In sum, our research provides a comprehensive understanding of several fundamental DICER mechanisms, challenging the long-standing model of the loop counting rule. This newfound knowledge reshapes our understanding of DICER's mechanisms, providing a robust foundation for future studies investigating the vast number of DICER mutations linked to various diseases.

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Project description:In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are central to sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation (RdDM) pathway. Here, we determined structure of a DCL3-pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated-A1 of the guide strand and the 1-nt 3′-overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated-end of the guide RNA and dual cleavage sites formed by the paired RNaseIII domains. These structural studies, complemented by functional data, reveal insights into the dicing principle for Dicers in general.

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