Abstract
Key message
Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allen RS, Li J, Stahle MI, DubroueA Gubler F, Millar AA (2007) Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family. P Natl Acad Sci USA 104:16371–16376
Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Brenner JL, Jasiewicz KL, Fahley AF, Kemp BJ, Abbott AL (2010) Loss of individual microRNAs causes mutant phenotypes in sensitized genetic backgrounds in C. elegans. Curr Biol 20:1321–1325
Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190
Bryant B, Macdonald W, Raikhel AS (2010) microRNA miR-275 is indispensable for blood digestion and egg development in the mosquito Aedes aegypti. P Natl Acad Sci USA 107:22391–22398
Chen XM (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucl Acids Res 33:e179
Chuck G, Candela H, Hake S (2009) Big impacts by small RNAs in plant development. Curr Opin Plant Biol 12:81–86
Davis S, Propp S, Freier SM, Jones LE, Serra MJ, Kinberger G, Bhat B, Swayze EE, Bennett CF, Esau C (2009) Potent inhibition of microRNA in vivo without degradation. Nucl Acids Res 37:70–77
Eamens AL, Agius C, Smith NA, Waterhouse PM, Wang MB (2011) Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana. Mol Plant 4:157–170
Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S, Lindholm M, Hedtjarn M, Hansen HF, Berger U et al (2008a) LNA-mediated microRNA silencing in non-human primates. Nature 452:896–897
Elmen J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A, Hedtjarn M, Hansen JB, Hansen HF, Straarup EM et al (2008b) Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucl Acids Res 36:1153–1162
Esau CC (2008) Inhibition of microRNA with antisense oligonucleotides. Methods 44:55–60
Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I, Leyva A, Weigel D, Garcia JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39:1033–1037
Gutierrez L, Bussell JD, Pacurar DI, Schwambach J, Pacurar M, Bellini C (2009) Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell 21:3119–3132
Horwich MD, Zamore PD (2008) Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells. Nat Protoc 3:1537–1549
Hutvagner G, Simard MJ, Mello CC, Zamore PD (2004) Sequence-specific inhibition of small RNA function. PLoS Biol 2:E98
Inui M, Martello G, Piccolo S (2010) MicroRNA control of signal transduction. Nature reviews. Mol Cell Biol 11:252–263
Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438:685–689
Leaman D, Chen PY, Fak J, Yalcin A, Pearce M, Unnerstall U, Marks DS, Sander C, Tuschl T, Gaul U (2005) Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121:1097–1108
Lennox KA, Behlke MA (2010) A direct comparison of anti-microRNA oligonucleotide potency. Pharm Res 27:1788–1799
Lennox KA, Behlke MA (2011) Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther 18:1111–1120
Li YF, Zheng Y, Addo-Quaye C, Zhang L, Saini A, Jagadeeswaran G, Axtell MJ, Zhang W, Sunkar R (2010) Transcriptome-wide identification of microRNA targets in rice. Plant J 62:742–759
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408
Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056
Mallory AC, Dugas DV, Bartel DP, Bartel B (2004) MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Curr Biol 14:1035–1046
Martin RC, Liu PP, Goloviznina NA, Nonogaki H (2010) microRNA, seeds, and Darwin?: diverse function of miRNA in seed biology and plant responses to stress. J Exp Bot 61:2229–2234
McGlinn E, Yekta S, Mansfield JH, Soutschek J, Bartel DP, Tabin CJ (2009) In ovo application of antagomiRs indicates a role for miR-196 in patterning the chick axial skeleton through Hox gene regulation. P Natl Acad Sci USA 106:18610–18615
Miska EA, Alvarez-Saavedra E, Abbott AL, Lau NC, Hellman AB, McGonagle SM, Bartel D, Ambros V, Horvitz HR (2007) Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. PLoS Genet 3:e215
Meister G, Landthaler M, Dorsett Y, Tuschl T (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10:544–550
Meng Y, Huang F, Shi Q, Cao J, Chen D, Zhang J, Ni J, Wu P, Chen M (2009) Genome-wide survey of rice microRNAs and microRNA-target pairs in the root of a novel auxin-resistant mutant. Planta 230:883–898
Meng Y, Ma X, Chen D, Wu P, Chen M (2010) MicroRNA-mediated signaling involved in plant root development. Biochem Biophys Res Commun 393:345–349
Obad S, dos Santos CO, Petri A, Heidenblad M, Broom O, Ruse C, Fu C, Lindow M, Stenvang J, Straarup EM et al (2011) Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet 43:371–378
Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133
Sieber P, Wellmer F, Gheyselinck J, Riechmann JL, Meyerowitz EM (2007) Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness. Development 134:1051–1060
Sun C, Hoglund AS, Olsson H, Mangelsen E, Jansson C (2005) Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling. Plant J 44:128–138
Sun C, Ridderstrale K, Hoglund AS, Larsson LG, Jansson C (2007) Sweet delivery—sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells. Plant J 52:1192–1198
Sun C, Ghebramedhin H, Hoglund AS, Jansson C (2008) Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology. Plant Signal Behav 3:328–330
Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Semin Cell Dev Biol 21:805–811
Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of MicroRNAs from rice. Plant Cell 17:1397–1411
Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17:49–63
Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D (2010) A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet 6:e1001031
Torres AG, Fabani MM, Vigorito E, Gait MJ (2011) MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection. RNA 17:933–943
Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12
Warthmann N, Chen H, Ossowski S, Weigel D, Herve P (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS One 3:e1829
Wu CI, Shen Y, Tang T (2009a) Evolution under canalization and the dual roles of microRNAs: a hypothesis. Genome Res 19:734–743
Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y (2009b) Rice MicroRNA effector complexes and targets. Plant Cell 21:3421–3435
Yan J, Gu Y, Jia X, Kang W, Pan S, Tang X, Chen X, Tang G (2012) Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24:415–427
Yoshida S, DAF, Cock JH, Gomez KA (1976) Laboratory manual for physiological studies of rice. 3rd ed
Zhou L, Liu Y, Liu Z, Kong D, Duan M, Luo L (2010) Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. J Exp Bot 61:4157–4168
Acknowledgments
The authors thank Chung-I Wu and Haijun Wen for participating in preliminary discussions on the development of the AMO technique and Yang Shen and Yang Lyu for critical reading of the manuscript. This work was supported by the National Science Foundation of China (91231117, 31170308, 31130069 and 41130208) and the Guangdong Outstanding Young Teachers Program.
Author contribution statement
LH, MX, JH and TZ carried out the experiments. LH, MX and TT wrote the manuscript. SS and TT designed the experiments. All authors read and approved the final manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Communicated by Z. Zhang.
L. He and M. Xie contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
He, L., Xie, M., Huang, J. et al. Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo. Plant Cell Rep 35, 933–945 (2016). https://doi.org/10.1007/s00299-016-1933-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-016-1933-y