Determinants of selectivity in the dicing mechanism III

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. small RNA sequencing

核糖核酸酶III（RNase III）DICER酶可通过切割前体分子（如前体微小RNA（pre-miRNA）、短发夹RNA（shRNA）及长双链RNA），在微小RNA（miRNA）和小干扰RNA（siRNA）等小RNA的生成过程中发挥关键作用。本研究采用高通量（High-throughput, HT）切割实验，深入探究了DICER的切割活性。我们对既往研究中广泛认可的2 nt环计数规则提出了质疑，并揭示了一种全新的作用机制——双分区碱基配对规则（bipartite base pairing rule）。该规则通过核糖核酸酶III结构域调控DICER的切割位点。此外，本研究阐明了此前已鉴定的YCR基序的识别机制。基于上述研究结果，我们还发现了一个可影响DICER切割位点的次级YCR基序。本研究同时解决了长期存在争议的、关于长茎RNA（如前体小干扰RNA（pre-siRNA）、长shRNA/pre-miRNA）上DICER切割位点的相关问题。研究证实，双链RNA结合结构域（double-stranded RNA binding domain, dsRBD）在决定长茎RNA中DICER切割位点的过程中发挥关键作用。综上，本研究全面阐明了DICER的多项核心作用机制，对长期沿用的环计数规则模型提出了挑战。这一新的研究成果重塑了我们对DICER作用机制的认知，为后续探究大量与多种疾病相关的DICER突变体的研究提供了坚实的理论基础。小RNA测序（small RNA sequencing）