MapToCleave: High-throughput profiling of microRNA biogenesis in living cells
Wenjing Kang, Bastian Fromm, Anna J.S. Houben, Eirik Høye, Daniela Bezdan, Carme Arnan, Kim Thrane, Michaela Asp, Rory Johnson, Inna Biryukova, Marc R. Friedländer
Abstract
Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.