Abstract
Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 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 we 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. We discovered that ‘slicer-dependency’ for the unwinding was affected primarily by certain parameters such as temperature and Mg2+. We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila.
Highlights
Small RNA-mediated silencing pathways are essential mechanisms that regulate gene expression in many eukaryotic organisms
Given that small RNAs are loaded into AGO proteins as double strands, the strand selection is already determined by the polarity of small RNA duplexes upon RNAinduced silencing complex (RISC) loading prior to the duplex unwinding [2,9]
We first established a cell-free system derived from human embryonic kidney (HEK) 293 cells expressing human AGO2 [21,22,32]
Summary
Small RNA-mediated silencing pathways are essential mechanisms that regulate gene expression in many eukaryotic organisms. Small RNA duplexes are actively incorporated into AGO proteins to form pre-RISCs, which requires the Hsc70–Hsp chaperone machinery [3,4,5,6]. This machinery, which is fueled by ATP hydrolysis, drives a dynamic conformational change in the AGO proteins, thereby allowing them to accommodate the duplex [3,4,5,6]. Given that small RNAs are loaded into AGO proteins as double strands, the strand selection is already determined by the polarity of small RNA duplexes upon RISC loading prior to the duplex unwinding [2,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
