Synergistic and Independent Actions of Multiple Terminal Nucleotidyl Transferases in the 3’ Tailing of Small RNAs in Arabidopsis
April
Synergistic and Independent Actions of Multiple Terminal Nucleotidyl Transferases in the 3' Tailing of Small RNAs in Arabidopsis
Xiaoyan Wang 0 1 2 3
Shuxin Zhang 0 1 2 3
Yongchao Dou 0 1 2 3
Chi Zhang 0 1 2 3
Xuemei Chen 0 1 2 3
Bin Yu 0 1 2 3
Guodong Ren 0 1 2 3
0 Funding: This work was supported by a grant from the National Natural Science Foundation of China (31471221) to GR (http://www.nsfc.gov.cn), a Startup Grant from Fudan University to GR (http://fudan.edu. cn), the National Science Foundation (MCB-1121193) to BY (http://www.nsf.gov), a Startup Grant from University of Nebraska-Lincoln to CZ (http://www.unl. edu), and the National Institutes of Health (GM061146) to XC (http://www.nih.gov). The funders
1 Data Availability Statement: All small RNA sequencing data are available from the GEO database (accession number GSE60826)
2 Editor: Michael J. Axtell, Pennsylvania State University , UNITED STATES
3 1 State Key Laboratory of Genetic Engineering, Institute of Plant Biology and Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai , China , 2 Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 3 Department of Botany and Plant Sciences & Institute of Integrative Genome Biology, University of California, Riverside, Riverside, California, United States of America, 4 Howard Hughes Medical Institute, University of California, Riverside, Riverside, California, United States of America, 5 Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University , Shanghai , China
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'-Omethylation. 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.
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Competing Interests: The authors have declared
that no competing interests exist.
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.
MicroRNAs (miRNAs), a class of small non-coding RNAs with 2024 nt in size, are master
regulators of gene expression at post-transcriptional levels in both plants and animals [1,2].
They impact various biological processes such as development, metabolism and response to
different biotic and abiotic stresses [1]. In Arabidopsis, miRNAs are typically derived from
their precursor RNAs called primary miRNA transcripts (pri-miRNAs) through a sequential
cleavage, from either loop proximal or loop distal, by DICER-LIKE 1 (DCL1) and its associated
pro (...truncated)