Flexible pri-miRNA structures enable tunable production of 5’ isomiRs

Abstract

Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5' isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that, in addition to previously known features, the overall structural flexibility of pri-miRNA impacts Drosha cleavage fidelity. Internal loops and nearby G-U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5' isomiR production. By analyzing patient data deposited in The Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5' isomiR biogenesis in tumors.