Molecular Mechanisms of MicroRNA Maturation and Destabilization in Human Argonautes

Abstract

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (Ago 1–4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that slicer-dependent unwinding via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the Ago2-RISC, here I show by careful re-examination that slicer-independent unwinding plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. I showed that in human cells, the elevated physiological temperature and the functional L1-PAZ domain of the Ago proteins, drive small RNA strand separation, even when slicer-assisted pathway is absent. In contrast to the previous models, I found that slicer-deficient Ago proteins can also be programmed with highly complementary siRNAs at the physiological temperature of humans, which now clearly explains why both miRNA and siRNA are found in all four human Ago proteins. Little is known about the regulation of miRNA stability. Mature miRNAs are stabilized by binding to Ago proteins, the core components of the RISC. Recent studies suggest that interactions between miRNAs and their highly complementary target RNAs promote release of miRNAs from Ago proteins, and this in turn can lead to destabilization of miRNAs. However, the physiological triggers of miRNA destabilization with molecular mechanisms remain largely unknown. Here, using an in vitro system that consists of a minimal human Ago2-RISC in HEK293T cell lysates, I sought to understand how miRNAs are destabilized by their targets. Strikingly, I showed that miRNA destabilization is dramatically enhanced by an interaction with seedless, non-canonical targets. I then showed that this process entails not only unloading of miRNAs from Ago, but also 3ʹ end destabilization of miRNAs occurred within Ago. Furthermore, mutation analysis suggests that conformational changes in the hinge region of the Ago PAZ domain are likely to be the main driving force of the miRNA destabilization. These collective results suggest that non-canonical targets may provide a stability control mechanism in the regulation of miRNAs in humans and also highlight the remarkable flexibility of human Ago proteins that experience dynamic conformational changes during their catalytic cycle. In sum, my findings reflect another layer onto the mutual regulatory circuits between Ago proteins, miRNAs and their targets, which may provide a means to fine-tune, refine and diversify miRNAs in cells.