PBOX-sRNA-seq uncovers novel features of miRNA modification and identifies selected 5'-tRNA fragments bearing 2'-O-modification.

The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3' end 2'-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2'-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2'-O-methylation. We also identified two highly conserved types of 5'-tRNA fragments (tRF) bearing HEN1-independent 2'-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.

Rapid growth of Moso bamboo (Phyllostachys edulis): Cellular roadmaps, transcriptome dynamics, and environmental factors.

Moso bamboo (Phyllostachys edulis) shows remarkably rapid growth (114.5 cm/day), but the underlying biological mechanisms remain unclear. After examining more than 12,750 internodes from more than 510 culms from 17 Moso populations, we identified internode 18 as a representative internode for rapid growth. This internode includes a 2-cm cell division zone (DZ), a cell elongation zone up to 12 cm, and a secondary cell wall (SCW) thickening zone. These zones elongated 11.8 cm, produced approximately 570,000,000 cells, and deposited ∼28 mg g-1 dry weight (DW) lignin and ∼44 mg g-1 DW cellulose daily, far exceeding vegetative growth observed in other plants. We used anatomical, mathematical, physiological, and genomic data to characterize development and transcriptional networks during rapid growth in internode 18. Our results suggest that (1) gibberellin may directly trigger the rapid growth of Moso shoots, (2) decreased cytokinin and increased auxin accumulation may trigger cell DZ elongation, and (3) abscisic acid and mechanical pressure may stimulate rapid SCW thickening via MYB83L. We conclude that internode length involves a possible tradeoff mediated by mechanical pressure caused by rapid growth, possibly influenced by environmental temperature and regulated by genes related to cell division and elongation. Our results provide insight into the rapid growth of Moso bamboo.

Uridylation and the SKI complex orchestrate the Calvin cycle of photosynthesis through RNA surveillance of TKL1 in Arabidopsis.

RNA uridylation, catalyzed by terminal uridylyl transferases (TUTases), represents a conserved and widespread posttranscriptional RNA modification in eukaryotes that affects RNA metabolism. In plants, several TUTases, including HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1), have been characterized through genetic and biochemical approaches. However, little is known about their physiological significance during plant development. Here, we show that HESO1 and URT1 act cooperatively with the cytoplasmic 3'-5' exoribonucleolytic machinery component SUPERKILLER 2 (SKI2) to regulate photosynthesis through RNA surveillance of the Calvin cycle gene TRANSKETOLASE 1 (TKL1) in Arabidopsis. Simultaneous dysfunction of HESO1, URT1, and SKI2 resulted in leaf etiolation and reduced photosynthetic efficiency. In addition, we detected massive illegitimate short interfering RNAs (siRNAs) from the TKL1 locus in heso1 urt1 ski2, accompanied by reduced TKL1/2 expression and attenuated TKL activities. Consequently, the metabolic analysis revealed that the abundance of many Calvin cycle intermediates is dramatically disturbed in heso1 urt1 ski2. Importantly, all these molecular and physiological defects were largely rescued by the loss-of-function mutation in RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), demonstrating illegitimate siRNA-mediated TKL silencing. Taken together, our results suggest that HESO1- and URT1-mediated RNA uridylation connects to the cytoplasmic RNA degradation pathway for RNA surveillance, which is crucial for TKL expression and photosynthesis in Arabidopsis.

A Josephson junction-coupled neuron with double capacitive membranes.

The channel currents have distinct magnetic field effect and any changes of the electromagnetic field or electirc stimulus will change the membrane potential effectively. A feasible neuron model considers the distinct physical characteristic is more suitable to mimic the neural activities accompanying with shift in energy level. A Josephson junction (JJ) is connected to a neural circuit for estimating the effect of external magnetic field and two capacitors are connected via a linear resistor for mimicing the capacitive field beside two sides of the cell membrane. Its equivalent Hamilton energy is calculated to show the relation between firing mode and energy level. Noisy disturbance is imposed to predict the occurrence of coherence resonance, and the biophysical neuron is excited to present higher energy level. This new neuron model can address the field effect and the biophysical property of cell membrane considered as combination of capacitive fields in double capacitors. It can mimic the physical property of outer and inner membranes, and energy exchange across the double membranes explains the energy mechanism in neural activities. Time-varying energy diveristy between capacitive field is crucial for supporting continuous firing activities. The JJ channel discerns slight changes in external magnetic field and regularity is stabilized under coherence resonance in presence of noisy excitation on the membrane or ion channels.

Molecular mechanism underlying the di-uridylation activity of Arabidopsis TUTase URT1.

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5'-AAAU-3' RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3' extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an 'open' to a 'closed' state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.

Wave propagation in a light-temperature neural network under adaptive local energy balance.

External electric and mechanical stimuli can induce shape deformation in excitable media because of its intrinsic flexible property. When the signals propagation in the media is described by a neural network, creation of heterogeneity or defect is considered as the effect of shape deformation due to accumulation or release of energy in the media. In this paper, a temperature-light sensitive neuron model is developed from a nonlinear circuit composed of a phototube and a thermistor, and the physical energy is kept in capacitive and inductive terms. Furthermore, the Hamilton energy for this function neuron is obtained in theoretical way. A regular neural network is built on a square array by activating electric synapse between adjacent neurons, and a few of neurons in local area is excited by noisy disturbance, which induces local energy diversity, and continuous coupling enables energy propagation and diffusion. Initially, the Hamilton energy function for a temperature-light sensitive neuron can be obtained. Then, the finite neurons are applied noise to obtain energy diversity to explore the energy spread between neurons in the network. For keeping local energy balance, one intrinsic parameter is regulated adaptively until energy diversity in this local area is decreased greatly. Regular pattern formation indicates that local energy balance creates heterogeneity or defects and a few of neurons show continuous parameter shift for keeping energy balance in a local area, which supports gradient energy distribution for propagating waves in the network.

Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack.

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

Spontaneous Seed Formation during Electrodeposition Drives Epitaxial Growth of Metastable Bismuth Selenide Microcrystals.

Materials with metastable phases can exhibit vastly different properties from their thermodynamically favored counterparts. Methods to synthesize metastable phases without the need for high-temperature or high-pressure conditions would facilitate their widespread use. We report on the electrochemical growth of microcrystals of bismuth selenide, Bi2Se3, in the metastable orthorhombic phase at room temperature in aqueous solution. Rather than direct epitaxy with the growth substrate, the spontaneous formation of a seed layer containing nanocrystals of cubic BiSe enforces the metastable phase. We first used single-crystal silicon substrates with a range of resistivities and different orientations to identify the conditions needed to produce the metastable phase. When the applied potential during electrochemical growth is positive of the reduction potential of Bi3+, an initial, Bi-rich seed layer forms. Electron microscopy imaging and diffraction reveal that the seed layer consists of nanocrystals of cubic BiSe embedded within an amorphous matrix of Bi and Se. Using density functional theory calculations, we show that epitaxial matching between cubic BiSe and orthorhombic Bi2Se3 can help stabilize the metastable orthorhombic phase over the thermodynamically stable rhombohedral phase. The spontaneous formation of the seed layer enables us to grow orthorhombic Bi2Se3 on a variety of substrates including single-crystal silicon with different orientations, polycrystalline fluorine-doped tin oxide, and polycrystalline gold. The ability to stabilize the metastable phase through room-temperature electrodeposition in aqueous solution without requiring a single-crystal substrate broadens the range of applications for this semiconductor in optoelectronic and electrochemical devices.

Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis.

During RNA-directed DNA methylation (RdDM), the DDR complex, composed of DRD1, DMS3, and RDM1, is responsible for recruiting DNA polymerase V (Pol V) to silence transposable elements (TEs) in plants. However, how the DDR complex is regulated remains unexplored. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) regulates the assembly of the DDR complex by targeting DMS3 for degradation. We found that a substantial set of RdDM loci was commonly de-repressed in apc/c and pol v mutants, and that the defects in RdDM activity resulted from up-regulated DMS3 protein levels, which finally caused reduced Pol V recruitment. DMS3 was ubiquitinated by APC/C for degradation in a D box-dependent manner. Competitive binding assays and gel filtration analyses showed that a proper level of DMS3 is critical for the assembly of the DDR complex. Consistent with the importance of the level of DMS3, overaccumulation of DMS3 caused defective RdDM activity, phenocopying the apc/c and dms3 mutants. Moreover, DMS3 is expressed in a cell cycle-dependent manner. Collectively, these findings provide direct evidence as to how the assembly of the DDR complex is regulated and uncover a safeguarding role of APC/C in the regulation of RdDM activity.

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3' to 5' exoribonuclease Atrimmer 2 in Arabidopsis.

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1 Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.

The NPR1-WRKY46-WRKY6 signaling cascade mediates probenazole/salicylic acid-elicited leaf senescence in Arabidopsis thaliana.

Endogenous salicylic acid (SA) regulates leaf senescence, but the underlying mechanism remains largely unexplored. The exogenous application of SA to living plants is not efficient for inducing leaf senescence. By taking advantage of probenazole (PBZ)-induced biosynthesis of endogenous SA, we previously established a chemical inducible leaf senescence system that depends on SA biosynthesis and its core signaling receptor NPR1 in Arabidopsis thaliana. Here, using this system, we identified WRKY46 and WRKY6 as key components of the transcriptional machinery downstream of NPR1 signaling. Upon PBZ treatment, the wrky46 mutant exhibited significantly delayed leaf senescence. We demonstrate that NPR1 is essential for PBZ/SA-induced WRKY46 activation, whereas WRKY46 in turn enhances NPR1 expression. WRKY46 interacts with NPR1 in the nucleus, binding to the W-box of the WRKY6 promoter to induce its expression in response to SA signaling. Dysfunction of WRKY6 abolished PBZ-induced leaf senescence, while overexpression of WRKY6 was sufficient to accelerate leaf senescence even under normal growth conditions, suggesting that WRKY6 may serve as an integration node of multiple leaf senescence signaling pathways. Taken together, these findings reveal that the NPR1-WRKY46-WRKY6 signaling cascade plays a critical role in PBZ/SA-mediated leaf senescence in Arabidopsis.

STV1, a ribosomal protein, binds primary microRNA transcripts to promote their interaction with the processing complex in Arabidopsis.

MicroRNAs (miRNAs) are key regulators of gene expression. They are processed from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by DNA-dependent polymerase II (Pol II). miRNA levels are precisely controlled to maintain various biological processes. Here, we report that SHORT VALVE 1 (STV1), a conserved ribosomal protein, acts in miRNA biogenesis in Arabidopsis A portion of STV1 localizes in the nucleus and binds pri-miRNAs. Using pri-miR172b as a reporter, we show that STV1 binds the stem-loop flanked by a short 5' arm within pri-miRNAs. Lack of STV1 reduces the association of pri-miRNAs with their processing complex. These data suggest that STV1 promotes miRNA biogenesis through facilitating the recruitment of pri-miRNAs to their processing complex. Furthermore, we show that STV1 indirectly involves in the occupancy of Pol II at the promoters of miRNA coding genes (MIR) and influences MIR promoter activities. Based on these results, we propose that STV1 refines the accumulation of miRNAs through its combined effects on pri-miRNA processing and transcription. This study uncovers an extraribosomal function of STV1.

The C-terminal cysteine-rich motif of NYE1/SGR1 is indispensable for its function in chlorophyll degradation in Arabidopsis.

KEY MESSAGE: The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential. During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.

DAWDLE Interacts with DICER-LIKE Proteins to Mediate Small RNA Biogenesis.

DAWDLE (DDL) is a conserved forkhead-associated (FHA) domain-containing protein with essential roles in plant development and immunity. It acts in the biogenesis of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), which regulate gene expression at the transcriptional and/or posttranscriptional levels. However, the functional mechanism of DDL and its impact on exogenous siRNAs remain elusive. Here, we report that DDL is required for the biogenesis of siRNAs derived from sense transgenes and inverted-repeat transgenes. Furthermore, we show that a mutation in the FHA domain of DDL disrupts the interaction of DDL with DICER-LIKE1 (DCL1), which is the enzyme that catalyzes miRNA maturation from primary miRNA transcripts (pri-miRNAs), resulting in impaired pri-miRNA processing. Moreover, we demonstrate that DDL interacts with DCL3, which is a DCL1 homolog responsible for siRNA production, and this interaction is crucial for optimal DCL3 activity. These results reveal that the interaction of DDL with DCLs is required for the biogenesis of miRNAs and siRNAs in Arabidopsis (Arabidopsis thaliana).

Quadruply-labeled serum albumin as a biodegradable nanosensor for simultaneous fluorescence imaging of intracellular pH values, oxygen and temperature.

The construction of multiple fluorescent nanosensors for intracellular studies is a challenging task because spectral overlap of indicator probes can lead to cross-talk and mutual interference. This work describes biodegradable nanosensors that can simultaneously measure three intracellular parameters (temperature, pH and oxygen concentration). Bovine serum albumin (BSA) is selected as the scaffold to construct the triple nanosensor by covalent immobilization four fluorophores on BSA. The following luminophores were used: (a) fluorescein as a probe for pH values, (b) a platinum(II) porphyrin complex for oxygen; (c) a europium(III) clathrate complex for temperature, and (d) a rhodamine B as a reference dye. The nanoparticles have a size of 20 nm and show excellent biocompatibility and good brightness. The nanosensors were used for ratiometric imaging of intracellular pH values, oxygen and temperature in HeLa cells. The triple nanosensor responds reversibly and this can be used for real-time tracing of these key parameters. Owing to their biodegradable feature, the use of this kind of triple nanosensor reduce the stress on cellular activities because less nanosensors can be used to gather the total information. Graphical abstractA triple nanosensor for simultaneously ratiometrically sensing intracellular pH, oxygen and temperature values was constructed by covalently labelling four fluorophores on a single serum albumin protein. The nanosensor shows good sensitivity, biocompatibility, is biodegradable and suitable for continuously measuring these important parameters.

The F-box protein HAWAIIAN SKIRT is required for mimicry target-induced microRNA degradation in Arabidopsis.

Mimicry target-directed microRNA degradation is widespread and highly conserved among eukaryotes. However, little is known about its mechanism of action. In this letter, by using STTM160 (target mimic of miR160) as a reporter, we show that dysfunction of HAWAIIAN SKIRT (HWS) suppresses the pleiotropic phenotype of STTM160. Small RNA sequencing and Northern blot analyses suggested that HWS only affects a subset of microRNAs. Intriguingly, we identified a stable coexistence of miR160/miR399 and their mimicry targets within the AGO1 complex when HWS is compromised, pointing to a possible role of HWS in the clearance of RNA-induced silencing complexes associated with mimicry target.

REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes.

The H3K27 methyltransferase CLF inhibits lateral root (LR) formation through depositing the repressive H3K27me3 mark to the chromatin of PIN1, a key polar auxin transporter gene. Here, we show that the H3K27me3 demethylase REF6 promotes lateral root primordium initiation and LR emergence. REF6 directly binds to the chromatin of PIN1/3/7. Dysfunction in REF6 results in increased levels of H3K27me3 on PIN1/3/7 and suppressed expression of PIN genes. Genetic analysis of the clf ref6 double mutant revealed an antagonistic action between CLF and REF6, in terms of LR formation. Our findings indicate that H3K27 methylation and demethylation activities are likely coordinated to ensure proper LR organogenesis.

NYEs/SGRs-mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis.

In the seed industry, chlorophyll (Chl) fluorescence is often used as a major non-invasive reporter of seed maturation and quality. Breakdown of Chl is a proactive process during the late stage of seed maturation, as well as during leaf senescence and fruit ripening. However, the biological significance of this process is still unclear. NYE1 and NYE2 are Mg-dechelatases, catalyzing the first rate-limiting step of Chl a degradation. Loss-of-function of both NYE1 and NYE2 not only results in a nearly complete retention of Chl during leaf senescence, but also produces green seeds in Arabidopsis. In this study, we showed that Chl retention in the nye1 nye2 double-mutant caused severe photo-damage to maturing seeds. Upon prolonged light exposure, green seeds of nye1 nye2 gradually bleached out and eventually lost their germination capacity. This organ-specific photosensitive phenotype is likely due to an over-accumulation of free Chl, which possesses photosensitizing properties and causes a burst of reactive oxygen species upon light exposure. As expected, a similar, albeit much milder, photosensitive phenotype was observed in the seeds of d1 d2, a green-seed mutant defective in NYE/SGR orthologous genes in soybean. Taken together, our data suggest that efficient NYEs-mediated Chl degradation is critical for detoxification during seed maturation.

Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis.

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.