Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing
![](/sites/rnajc.ucsf.edu/files/styles/large/public/Image-Categories/RNA-Structure%2C-Folding-%26-Modeling/80713888-education-chart-of-biology-for-rna-structure-diagram.jpg?itok=6P2J3MER)
Abstract
To use microRNAs to downregulate mRNA targets, cells must first process these 22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.