Single Molecule Observation of MicroRNA Processing Enzymes at Action

Abstract

MicroRNAs (miRNAs) regulate mRNA expression via RNA interference. MicroRNA is generated when nuclear RNase III Drosha cleaves a long primary transcript liberating a hairpin precursor miRNA (pre-miRNA). The pre-miRNA is processed into a ∼22 nt mature miRNA by cytoplasmic RNase III Dicer. In stem cells and certain cancer cells, the maturation process is interrupted when the pre-miRNA is hijacked by Lin28, a pluripotency factor, and the 3′ end of the RNA is uridylated by a noncanonical polyA polymerase, TUT4.With single molecule fluorescence microscopy, here we reveal the action mechanisms of human Dicer and TUT4 proteins at the molecular level. As it is difficult to prepare as large eukaryotic proteins as human Dicer and TUT4 proteins, we used a single molecule immunoprecipitation technique that we had recently developed [1].We, for the first time, observed Dicer's cleavage activity in real time using single molecule FRET and revealed stepwise processes of the cleavage action. Next, we investigated how Lin28 interfered with Dicer's activity and mediated TUT4's uridylation of pre-miRNA. Our real-time analysis suggests that Lin28 functions as a processivity factor of TUT4 and TUT4 utilizes a looping mechanism when it attaches uridines to a recruited pre-miRNA.Reference:[1] K. H. Yeom, I. Heo, J. Lee, S. Hohng, V. N. Kim, C. Joo, (2011) “Single Molecule Approach to Immunoprecipitated Protein Complexes: Insights into MiRNA Uridylation” EMBO Reports, 12, 690-696.