Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China.
Nat Commun. 2022 Apr 19;13(1):2138. doi: 10.1038/s41467-022-29822-3.
The accurate and efficient cleavage of shRNAs and pre-miRNAs by DICER is crucial for their gene-silencing activity. Here, we conduct high-throughput DICER cleavage assays for more than ~20,000 different shRNAs and show the comprehensive cleavage activities of DICER on these sequences. We discover a single-nucleotide bulge (22-bulge), which facilitates the cleavage activity of DICER on shRNAs and human pre-miRNAs. As a result, this 22-bulge enhances the gene-silencing activity of shRNAs and the accuracy of miRNA biogenesis. In addition, various single-nucleotide polymorphism-edited 22-bulges are found to govern the cleavage sites of DICER on pre-miRNAs and thereby control their functions. Finally, we identify the single cleavage of DICER and reveal its molecular mechanism. Our findings improve the understanding of the DICER cleavage mechanism, provide a foundation for the design of accurate and efficient shRNAs for gene-silencing, and indicate the function of bulges in regulating miRNA biogenesis.
DICER 对 shRNAs 和 pre-miRNAs 的精确和高效切割对于它们的基因沉默活性至关重要。在这里，我们进行了超过 20,000 种不同 shRNAs 的高通量 DICER 切割分析，并展示了 DICER 对这些序列的全面切割活性。我们发现了一个单核苷酸凸起（22 个凸起），这促进了 DICER 对 shRNAs 和人 pre-miRNAs 的切割活性。因此，这个 22 个凸起增强了 shRNAs 的基因沉默活性和 miRNA 生物发生的准确性。此外，还发现各种单核苷酸多态性编辑的 22 个凸起控制了 DICER 在 pre-miRNAs 上的切割位点，从而控制了它们的功能。最后，我们确定了 DICER 的单一切割，并揭示了其分子机制。我们的研究结果提高了对 DICER 切割机制的理解，为设计用于基因沉默的精确和高效 shRNAs 提供了基础，并表明了凸起在调节 miRNA 生物发生中的作用。
