Functional atlas of primary miRNA maturation by the Microprocessor

Primary microRNAs are the precursors of microRNAs which 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, distinguishing features of primary microRNA compared to other hairpin forming transcripts should be further explored. We developed a massively parallel functional assay termed Dro-seq that enabled testing of hundreds of known primary microRNA substrates and thousands of single nucleotide variants of well-known microRNAs. We found an additional feature of primary microRNAs, called Shannon entropy describing the structural ensemble is important for processing. In a deep mutagenesis experiment, we find particular apical loop U bases are likely recognized by DGCR8 and are important for efficient processing. These findings build on known about primary miRNA maturation by Microprocessor and further explore the substrate RNA sequence-structure relationship.