Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.
Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.
Mol Cell. 2020 Dec 3;80(5):892-902.e4. doi: 10.1016/j.molcel.2020.10.028. Epub 2020 Nov 13.
Primary microRNAs (miRNAs) are the precursors of miRNAs that modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, a complete understanding of what features distinguish primary miRNA from hairpin structures in other transcripts is still lacking. We develop a massively parallel functional assay termed Dro-seq (Drosha sequencing) that enables testing of hundreds of known primary miRNA substrates and thousands of single-nucleotide variants. We find an additional feature of primary miRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we observe particular apical loop U bases, likely recognized by DGCR8, are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by the Microprocessor and further explore the substrate RNA sequence-structure relationship.
初级 microRNAs (miRNAs) 是 miRNAs 的前体，可调节人类大多数 mRNAs 的表达。它们折叠成发夹结构，在其基部被称为微处理器（Drosha/DGCR8）的酶复合物切割。虽然已经了解了许多分子细节，但对于区分初级 miRNA 和其他转录本中的发夹结构的特征仍缺乏完整的认识。我们开发了一种大规模并行的功能测定方法，称为 Dro-seq（Drosha 测序），该方法可用于测试数百种已知的初级 miRNA 底物和数千种单核苷酸变体。我们发现了初级 miRNA 的另一个特征，称为香农熵，它描述了对加工很重要的结构整体。在一项深度诱变实验中，我们观察到可能被 DGCR8 识别的特定顶端环 U 碱基对于有效加工很重要。这些发现建立在 Microprocessor 对初级 miRNA 成熟的现有认识基础上，并进一步探索了底物 RNA 序列-结构关系。
