Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

Abstract

RNA interfering is a eukaryote-specific gene silencing by 20∼23-nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3′ end-shortened variants of let-7a, miR-27a, and specific miR-17–92 families that make AGO3 an extremely competent slicer, increasing target cleavage up to ∼82-fold in some instances. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

Are Argonaute-Associated Tiny RNAs Junk, Inferior miRNAs, or a New Type of Functional RNAs?

“…Such short RNAs would be quite susceptive to cellular RNases because they are too short to fold into a hairpin structure or form a stable duplex. Supporting this idea, my recent study showed that transfection of an unmodified, 14 nt guide RNA into HEK293T cells failed to activate AGO3 (Park et al, 2020), probably due to single-stranded RNA degradation by the cellular RNases (Lima et al, 2012). However, when the same 14 nt single-stand was heavily modified, it loaded onto AGO3 and converted it to an active slicer.…”

“…Therefore, few transcripts encompass the perfectly complementary sequence, which could explain why only a few mRNAs are cleaved by AGO2 with mature miRNAs. My recent study discovered that loading 14 nt specific tyRNA catalytically activates AGO3, while reducing the slicing activity of AGO2 compared to that of their mature miRNAs ( Park et al, 2020 ). The sequence fully complementary to a 14 nt cityRNA appears every 67 million nucleotides (= 4 13 = 67 × 10 6 ).…”

“…Assuming that about 93% of the human genome is transcribed ( Consortium et al, 2007 ), AGO3 loaded with a 14 nt cityRNA could cleave approximately 100 sites. This number decreases because among 14–15 nt tyRNAs, only ones with specific sequences can serve as cityRNAs (i.e., catalytically activate AGO3) ( Park et al, 2020 ). Nevertheless, the synthesis of cityRNAs appears to be a powerful tool for the cell to change gene expression drastically by cleaving many transcripts.…”

“…This review defines AGO-associated 19 nt or longer small RNAs as mature miRNAs, with those shorter than 19 nt as tyRNAs. Although these tyRNAs have been reported to retain a similar physiological activity to their mature miRNAs, some of 14–15 nt tyRNAs drastically increase the slicing activity of human AGO3 and named them cleavage-inducing tyRNAs (cityRNAs) ( Park et al, 2020 ). The discovery has raised the possibility that tyRNAs confer yet-unidentified, but distinct roles on AGOs.…”

Argonaute Proteins Take Center Stage in Cancers

“…In mammalian cells, AGO1–4 proteins mediate miRNA-dependent translational repression. Moreover, AGO2, and, to a limited extent, AGO3, possess endoribonuclease activity, which can catalyze smRNA-directed ribonucleolytic cleavage of target mRNAs [ 41 , 42 , 43 ].…”