Synergistic and Independent Actions of Multiple Terminal Nucleotidyl Transferases in the 3’ Tailing of Small RNAs in Arabidopsis

Abstract

All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3’ terminal 2’-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3’ uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3’-to-5’ trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3’ uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3’ tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3’ end modification and stability control.

The expanding world of small RNAs in plants

“…2). Recent studies have shown that HESO1 acts on most miRNAs, whereas the monouridylation of certain miRNAs requires another nucleotidyl transferase, UTP: RNA URIDYLYLTRANSFERASE (URT1) 53,54 . HESO1 seems to be more processive than URT1, perhaps because of their different substrate preference, which depends on the 3′ terminal nucleotide of the small RNAs: URT1 prefers 3′ adenine whereas HESO1 has strong preference for 3′ uridine, thus explaining why it polyuridylates its substrates.…”

“…Other types of miRNA tailings were found in mutants lacking both HESO1 and URT1 activities, including non-uridine nucleotides 53 , suggesting that other small RNA modification pathways might exist in plants. There are eight nucleotidyl transferases that remain uncharacterized in A. thaliana , and these could have a role in miRNA modification or in processes utilizing secondary or heterochromatic siRNAs.…”

“…Thus, the 3′ methylation of miRNAs loaded onto AGO1 serves to protect them from HESO1 activity. Recent studies have shown that URT1 also interacts with AGO1 to establish mono-uridylation of particular miRNAs 53,54 (left), and this process may produce 22-nucleotide miRNA variants that are able to form functional RNA-induced silencing complex es and trigger post-transcriptional gene silencing (PTGS) 54 (see Fig. 3).…”

Regulation of Plant miRNA Biogenesis

“…2'-O methylation protects miRNAs from both 3'-uridylation and 3'-truncations. miRNAs that are not 2'-O methylated by HEN1 are readily 3'-uridylated by HEN1 Suppressor 1 (HESO1) and UTP:RNA Uridylyltransferase 1 (URT1) (Tu et al, 2015;X. Wang et al, 2015) and gets degraded.…”

“…3'-poly-uridylation of AGO1 bound miRNA decreases its targeting efficiency. Both HESO1 and URT1 have similar functions but slightly varied substrate specificities and can act sequentially or co-operatively (Ren, Chen, et al, 2012;Zhao et al, 2012;Tu et al, 2015;X. Wang et al, 2015).…”