Functional Atlas of Primary miRNA Maturation by the Microprocessor.

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. • Microprocessor activity across miRBase is profiled in vitro using Dro-seq • High-throughput mutagenesis allows analysis at single-nucleotide resolution • A functional atlas of primary miRNA maturation by the Microprocessor is described • Pri-miRNAs with low Shannon entropy are superior substrates for Drosha Primary microRNAs processed by the Microprocessor are diverse in sequence and structure. Rice et al. developed Dro-seq to extensively profile the activity of the Microprocessor against known human miRNAs and thousands of variants. An analysis of RNA structure features showed that low Shannon entropy is essential for efficient cutting. [ABSTRACT FROM AUTHOR]