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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., Author Summary Small silencing RNAs are key regulators of gene expression in both plants and animals. HEN1-mediated 3’ terminal 2’-O-methylation plays a crucial role in small RNA stability control. In the absence of HEN1, several types of small RNAs become frequently uridylated (non-templated uridine addition) and trimmed, a phenomenon that is conserved across species. However, the underlying molecular mechanism is barely understood. In this study, we have discovered UTP: RNA uridylyltransferase (URT1) that acts synergistically with HESO1 in miRNA uridylation, in addition to its role in oligo-adenylated mRNA uridylation. Analyzing the miRNA profiles also reveals the existence of multiple terminal nucleotidyl transferases in the miRNA tailing process and an antagonistic action between uridylation and trimming. We believe this study will shed light on our understanding of how various terminal nucleotidyl transferases recognize their substrates and function coordinately.

3’ uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2’-O-methylation on the 3’ terminal ribose protects micro(mi)RNAs from 3’ truncation and 3’ uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3’ tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3’ end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3’ tailing could affect the activities of miRNAs in addition to leading to miRNA degradation., Author Summary The tailing of RNAs with non-templated uridines, known as uridylation, is often associated with RNA degradation. We previously identified HESO1 as a nucleotidyl transferase that uridylates microRNAs (miRNAs) to lead to their degradation in Arabidopsis. But HESO1 cannot account for all the miRNA uridylation activity in vivo. Here, we have uncovered UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) as another nucleotidyl transferase that uridylates miRNAs. HESO1 and URT1 have different substrate preferences and act cooperatively to tail miRNAs. We show that both enzymes are able to act on ARGONAUTE1 (AGO1)-bound miRNAs and that the tailed miRNAs stay bound by AGO1. We show that URT1-mediated tailing affects the activities of miR165/6 and miR171a differently. This study reveals intricate miRNA uridylation processes as well as functional outcomes of miRNA uridylation.