MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem-loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base-pairing throughout the remainder of the stem, and a narrow stem-length preference of 35±1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs, in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence, yet processed more efficiently than natural pri-miRNAs.
Overall design: Three major experiments are included in the submitted data. 1) Pools of synthetic DNA containing barcodes and pri-miRNA variants were sequenced to infer the barcode-pri-miRNA linkages, and the pri-miRNA variants were assayed for their cleavage efficiency ("selection"), which can be quantified by comparing their representations in the input and after cleavage. 2) Artificial miRNAs produced in HEK293T cells were sequenced. 3) Transcriptomes of artificial miRNA-expressed cells were sequenced to quantify mRNA changes.
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