Distinct and Cooperative Activities of HESO1 and URT1 Nucleotidyl Transferases in MicroRNA Turnover in Arabidopsis

PLoS Genetics, Apr 2015

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.

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Distinct and Cooperative Activities of HESO1 and URT1 Nucleotidyl Transferases in MicroRNA Turnover in Arabidopsis

April Distinct and Cooperative Activities of HESO1 and URT1 Nucleotidyl Transferases in MicroRNA Turnover in Arabidopsis Bin Tu 0 1 2 3 Li Liu 0 1 2 3 Chi Xu 0 1 2 3 Jixian Zhai 0 1 2 3 Shengben Li 0 1 2 3 Miguel A. Lopez 0 1 2 3 Yuanyuan Zhao 0 1 2 3 Yu Yu 0 1 2 3 Vanitharani Ramachandran 0 1 2 3 Guodong Ren 0 1 2 3 Bin Yu 0 1 2 3 Shigui Li 0 1 2 3 Blake C. Meyers 0 1 2 3 Beixin Mo 0 1 2 3 Xuemei Chen 0 1 2 3 0 1 Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, Riverside, California, United States of America, 2 Rice Research Institute, Sichuan Agricultural University , Chengdu Wenjiang, Sichuan , China , 3 Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University , Shenzhen , China , 4 Department of Plant & Soil Sciences, and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States of America, 5 Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska- Lincoln, Lincoln, Nebraska, United States of America, 6 Howard Hughes Medical Institute, University of California , Riverside, Riverside, California , United States of America 1 Data Availability Statement: All small RNA sequencing data are available from the GEO database (accession number GSE61362) 2 Editor: Michael J. Axtell, Pennsylvania State University UNITED STATES 3 a Current address: Monsanto Company, Chesterfield, Missouri, United States of America b Current address: State Key Laboratory of Genetic Engineering, Institute of Plant Biology and Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai , China 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 URIDY- - Funding: The work was supported by grants from NIH (GM061146) and Gordon and Betty Moore Foundation (GBMF3046) to XC, NSF (MCB-1121193) to BY, National Natural Science Foundation of China to XC (91440105) and to BM (31210103901 and 30970265), and USDA NIFA (2012-67013-19396) to BCM. BT was supported by a fellowship from China Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. LYLTRANSFERASE 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. Competing Interests: The authors have declared that no competing interests exist. 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. The three major types of small RNAs in eukaryotes, microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), impact many biological processes such as development, self/non-self recognition, genome stability, and adaption to environment. Given the widespread and indispensible functions of small RNAs, it is crucial to understand their biogenesis and turnover. A common step in the biogenesis of miRNAs and siRNAs in plants, as well as piRNAs and certain endogenous siRNA (...truncated)


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Bin Tu, Li Liu, Chi Xu, Jixian Zhai, Shengben Li, Miguel A. Lopez, Yuanyuan Zhao, Yu Yu, Vanitharani Ramachandran, Guodong Ren, Bin Yu, Shigui Li, Blake C. Meyers, Beixin Mo, Xuemei Chen. Distinct and Cooperative Activities of HESO1 and URT1 Nucleotidyl Transferases in MicroRNA Turnover in Arabidopsis, PLoS Genetics, 2015, 4, DOI: 10.1371/journal.pgen.1005119