Human Argonaute3 has slicer activity

Abstract

Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes that bind target mRNAs and silence gene expression through translational repression and deadenylation but not cleavage. Here, we report that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs, whereas AGO3 loaded with let-7a, miR-19b or miR-16 does not, indicating that AGO3 has slicer activity but that this activity depends on the guide RNA. Our cleavage assays using chimeric guides revealed the significance of seed sequence for AGO3 activity, which depends specifically on the sequence of the post-seed. Unlike AGO2, target cleavage by AGO3 requires both 5′- and 3′-flanking regions. Our 3.28 Å crystal structure shows that AGO3 forms a complete active site mirroring that of AGO2, but not a well-defined nucleic acid-binding channel. These results demonstrating that AGO3 also has slicer activity but with more intricate substrate requirements, explain the observation that AGO3 has retained the necessary catalytic residues throughout its evolution. In addition, our structure inspires the idea that the substrate-binding channel of AGO3 and consequently its cellular function, may be modulated by accessory proteins.

Noncanonical functions of microRNA pathway enzymes – Drosha, DGCR8, Dicer and Ago proteins

“…Among all four human Ago proteins, Ago1‐4, only Ago2 was thought to have slicer activity , which mediates endonucleolytic cleavage of mRNA . Recently, Ago3 has been reported to also possess slicer ability but such activity is substrate‐dependent . Ago proteins have extensive properties that are not limited to miRNA‐mediated RNAi in the cytoplasm (Fig.…”

A fungal Argonaute interferes with RNA interference

“…Target RNA cleavage assay was performed as described previously (16) with modifications. Slicer assays were performed in a reaction buffer containing 25 mM HEPES-KOH pH 7.5, 5 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol, 2 mM adenosine triphosphate, 0.05 mg/ml bovine serum albumin, 2 U/μl RiboLock RNase Inhibitor (Thermo Scientific).…”

AGO1 in association with NEAT1 lncRNA contributes to nuclear and 3D chromatin architecture in human cells

“…Additionally, our data show that the essential paraspeckle proteins specifically associate with AGO1 but not AGO2 nuclear complexes, indicating different functions for AGO1 and AGO2 in the nucleus. Importantly, in contrast to AGO2 (Meister et al 2004) and AGO3 (Park et al 2017), human AGO1 does not contain a functional slicing domain (Wu et al 2008). Thus its nuclear function appears to be independent of RNA-processing, but instead, it depends on RNA binding.…”