Abstract
Although much is known about microRNA (miRNA) biogenesis and the mechanism by which miRNAs regulate their targets, little is known about the regulation of miRNA stability. Mature miRNAs are stabilized by binding to Argonaute (Ago) proteins, the core components of the RNA-induced silencing complex (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, we sought to understand how miRNAs are destabilized by their targets. Strikingly, we showed that miRNA destabilization is dramatically enhanced by an interaction with seedless, non-canonical targets. We then showed that this process entails not only unloading of miRNAs from Ago, but also 3΄ end destabilization of miRNAs occurred within Ago. Furthermore, our mutation analysis indicates that conformational changes in the hinge region of the Ago PAZ domain are likely to be the main driving force of the miRNA destabilization. Our collective results suggest that non-canonical targets may provide a stability control mechanism in the regulation of miRNAs in humans.
Highlights
MicroRNAs are ∼22-nucleotide, small regulatory RNA molecules that play important roles in a wide range of biological processes. miRNAs are transcribed as primary miRNA transcripts that are processed via two cleavage steps that are mediated by Drosha and Dicer [1,2]
We hypothesized that if Ago proteins are degraded by proteases in cells, miRNAs should be rapidly degraded by nucleases
To test this idea in vitro, we immunopurified Ago2 complexes and subjected them to protease digestion, followed by RNase treatment (Figure 1A). miRNAs were degraded rapidly by RNases in the protease-treated control, whereas those in Ago2 complexes were largely protected from degradation, suggesting that Ago protein is required for mature miRNA stability (Figure 1A)
Summary
MicroRNAs (miRNAs) are ∼22-nucleotide (nt), small regulatory RNA molecules that play important roles in a wide range of biological processes. miRNAs are transcribed as primary miRNA (pri-miRNA) transcripts that are processed via two cleavage steps that are mediated by Drosha and Dicer [1,2]. MiRNAs are transcribed as primary miRNA (pri-miRNA) transcripts that are processed via two cleavage steps that are mediated by Drosha and Dicer [1,2]. These tandem actions convert pri-miRNAs into precursor miRNAs (pre-miRNAs) and results in the production of the ∼21–23 nt miRNA duplexes. The miRNA duplexes, which contain a 5 phosphate and a 2nt 3 overhang on each end, are subsequently loaded into Argonaute (Ago) proteins with the aid of chaperone machinery [3,4]. The two strands of the duplex are separated within the Ago proteins. The seed region (nt 2–8) of mature miRNAs directs the RNA-induced silencing complex (RISC) to target mRNAs by binding to complementary sequences [8], which results in mRNA destabilization and/or translational repression [9,10]
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