Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

Abstract

In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. S mall interfering RNAs (siRNAs) and microRNAs (miRNAs), ∼20-25 nucleotides (nt) in size, are important regulators of gene expression. miRNAs and siRNAs are derived from imperfect hairpin transcripts and perfect long double-stranded RNAs, respectively (1, 2). miRNAs and siRNAs are then associated with Argonaute (AGO) proteins to repress gene expression through target cleavage and/or translational inhibition (3). The cleavage of target mRNAs usually occurs at a position opposite the tenth and eleventh nucleotides of miRNAs, resulting in a 5′ RNA fragment (5′ fragment) and a 3′ fragment (4). In Arabidopsis, the major effector protein for miRNA-mediated gene silencing is AGO1, which possesses the endonuclease activity required for target cleavage (5-7). In Drosophila, the exosome removes the 5′ fragments through its 3′-to-5′ exoribonuclease activity (8). How 5′ fragments are degraded in higher plants remains unknown. It has been shown that the 5′ fragments are subject to untemplated uridine addition at their 3′ termini (uridylation) in both animals and plants (9). However, the biological significance of this modification remains unknown because of a lack of knowledge of the enzymes targeting 5′ fragments for uridylation.Uridylation plays important roles in regulating miRNA biogenesis. In animals, TUT4, a terminal uridyl transferase, is recruited by Lin-28 (an RNA binding protein) to the let-7 precursor (prelet-7), resulting in uridylation of prelet-7 (10, 11). This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in ani...

The Slicer Activity of ARGONAUTE1 is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis

“…This is consistent with trimming of the 39-overhang of the AGO1-bound guide strand in an siRNA duplex. AGO1-dependent 39-modifications, including trimming and uridylation, have previously been observed (Zhai et al, 2013;Ren et al, 2014), and it is possible that the trimming observed here is a manifestation of the same phenomenon that produces the previously described AGO1-dependent 39-trimming of small RNA. We could not clearly assign the faster migrating species of siRNAs other than siR255 to reads detected by deep sequencing.…”

Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function

“…In Arabidopsis, the RISC 39-cleaved fragment contains a free 59-monophosphate, making it a substrate for XRN4 (Souret et al, 2004). RISC 59-cleaved fragments undergo rapid turnover upon uridylation at their 39 end by the miRNA nucleotidyl transferase HEN1 SUPPRESSOR1 (Ren et al, 2014). The pathways participating in RISC 59-cleaved fragment demise could include 59-39 decay by XRN4 (Ren et al, 2014), although the mechanism of the prerequisite removal of the 59 cap from these fragments is largely unknown.…”

“…RISC 59-cleaved fragments undergo rapid turnover upon uridylation at their 39 end by the miRNA nucleotidyl transferase HEN1 SUPPRESSOR1 (Ren et al, 2014). The pathways participating in RISC 59-cleaved fragment demise could include 59-39 decay by XRN4 (Ren et al, 2014), although the mechanism of the prerequisite removal of the 59 cap from these fragments is largely unknown. Alternatively, the uridylated fragments may be degraded in the 39 to 59 direction by the exosome (see below).…”

Small RNAs meet their targets: When methylation defends miRNAs from uridylation

“…40 In a recent study, we show that HESO1 is responsible for the uridylation of 5 0 CPs in Arabidopsis. 41 A reduction in uridylation in heso1-2 increases the accumulation of 5 0 CP, demonstrating that uridylation triggers degradation of 5 0 CP. 41 The proportion of 3 'truncated 5 0 CPs is increased by heso1 relative to wild-type plants (WT), suggesting that uridylation may cause 5 0 CP degradation through a mechanism different from 3 0 -to-5 0 trimming.…”