Plants produce a diverse array of small RNA molecules capable of gene regulation, including Pol IV-dependent short interfering (p4-si)RNAs that trigger transcriptional gene silencing. Small RNA transcriptomes are available for many plant species, but mutations affecting the synthesis of Pol IV-dependent siRNAs are characterized only in Arabidopsis and maize, leading to assumptions regarding nature of p4-siRNAs in all other species. We have identified a mutation in the largest subunit of Pol IV, NRPD1, that impacts Pol IV activity in Brassica rapa, an agriculturally important relative of the reference plant Arabidopsis. Using this mutation we characterized the Pol IV-dependent and Pol IV-independent small RNA populations in B. rapa. In addition, our analysis demonstrates reduced production of p4-siRNAs in B. rapa relative to Arabidopsis. B. rapa genomic regions are less likely to generate p4-siRNAs than Arabidopsis but more likely to generate Pol IV-independent siRNAs, including 24 nt RNAs mapping to transposable elements. These observations underscore the diversity of small RNAs produced by plants and highlight the importance of genetic studies during small RNA analysis.
References
[1]
Valencia-Sanchez, M.A.; Liu, J.; Hannon, G.J.; Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Gene Dev. 2006, 20, 515–524, doi:10.1101/gad.1399806.
[2]
Moazed, D. Small RNAs in transcriptional gene silencing and genome defence. Nature 2009, 457, 413–420, doi:10.1038/nature07756.
[3]
Mosher, R.A.; Schwach, F.; Studholme, D.; Baulcombe, D.C. PolIVb influences RNA-directed DNA-methylation independently of its role in siRNA biogenesis. Proc. Natl. Acad. Sci. USA 2008, 105, 3145–3150, doi:10.1073/pnas.0709632105.
[4]
Haag, J.R.; Ream, T.S.; Marasco, M.; Nicora, C.D.; Norbeck, A.D.; Pasa-Tolic, L.; Pikaard, C.S. In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol. Cell 2012, 48, 811–818, doi:10.1016/j.molcel.2012.09.027.
[5]
Xie, Z.; Johansen, L.K.; Gustafson, A.M.; Kasschau, K.D.; Lellis, A.D.; Zilberman, D.; Jacobsen, S.E.; Carrington, J.C. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2004, 2, 642–652.
Zhang, X.; Henderson, I.R.; Lu, C.; Green, P.J.; Jacobsen, S.E. Role of RNA polymerase IV in plant small RNA metabolism. Proc. Natl. Acad. Sci. USA 2007, 104, 4536–4541.
[10]
Herr, A.J.; Jensen, M.B.; Dalmay, T.; Baulcombe, D. RNA polymerase IV directs silencing of endogenous DNA. Science 2005, 308, 118–120, doi:10.1126/science.1106910.
Onodera, Y.; Haag, J.R.; Ream, T.; Nunes, P.C.; Pontes, O.; Pikaard, C.S. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 2005, 120, 613–622, doi:10.1016/j.cell.2005.02.007.
[13]
Pontier, D.; Yahubyan, G.; Vega, D.; Bulski, A.; SaezVasquez, J.; Hakimi, M.-A.; Lerbs-Mache, S.; Colot, V.; Lagrange, T. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Gene Dev. 2005, 19, 2030–2040, doi:10.1101/gad.348405.
[14]
Chan, S.W.; Zilberman, D.; Xie, Z.; Johansen, L.K.; Carrington, J.C.; Jacobsen, S.E. RNA silencing genes control de novo DNA methylation. Science 2004, 303, 1336, doi:10.1126/science.1095989.
[15]
Perez-Hormaeche, J.; Potet, F.; Beauclair, L.; le Masson, I.; Courtial, B.; Bouche, N.; Lucas, H. Invasion of the Arabidopsis genome by the tobacco retrotransposon Tnt1 is controlled by reversible transcriptional gene silencing. Plant Physiol. 2008, 147, 1264–1278, doi:10.1104/pp.108.117846.
[16]
Mari-Ordonez, A.; Marchais, A.; Etcheverry, M.; Martin, A.; Colot, V.; Voinnet, O. Reconstructing de novo silencing of an active plant retrotransposon. Nat. Genet. 2013, 45, 1029–1039, doi:10.1038/ng.2703.
Pikaard, C.S.; Haag, J.R.; Ream, T.; Wierzbicki, A.T. Roles of RNA polymerase IV in gene silencing. Trends Plant Sci. 2008, 13, 390–397, doi:10.1016/j.tplants.2008.04.008.
[19]
Nobuta, K.; Lu, C.; Shrivastava, R.; Pillay, M.; de Paoli, E.; Accerbi, M.; Arteaga-Vazquez, M.; Sidorenko, L.; Jeong, D.H.; Yen, Y.; et al. Distinct size distribution of endogeneous siRNAs in maize: Evidence from deep sequencing in the mop1–1 mutant. Proc. Natl. Acad. Sci. USA 2008, 105, 14958–14963, doi:10.1073/pnas.0808066105.
[20]
Szittya, G.; Moxon, S.; Santos, D.M.; Jing, R.; Fevereiro, M.P.; Moulton, V.; Dalmay, T. High-throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families. BMC Genomics 2008, 9, 593, doi:10.1186/1471-2164-9-593.
[21]
Mohorianu, I.; Schwach, F.; Jing, R.; Lopez-Gomollon, S.; Moxon, S.; Szittya, G.; Sorefan, K.; Moulton, V.; Dalmay, T. Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns. Plant. J. 2011, 67, 232–246, doi:10.1111/j.1365-313X.2011.04586.x.
[22]
Jeong, D.H.; Park, S.; Zhai, J.; Gurazada, S.G.; de Paoli, E.; Meyers, B.C.; Green, P.J. Massive analysis of rice small RNAs: Mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage. Plant Cell 2011, 23, 4185–4207, doi:10.1105/tpc.111.089045.
[23]
Aryal, R.; Yang, X.; Yu, Q.; Sunkar, R.; Li, L.; Ming, R. Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya. BMC Genomics 2012, 13, 682, doi:10.1186/1471-2164-13-682.
[24]
Ortiz-Morea, F.A.; Vicentini, R.; Silva, G.F.; Silva, E.M.; Carrer, H.; Rodrigues, A.P.; Nogueira, F.T. Global analysis of the sugarcane microtranscriptome reveals a unique composition of small RNAs associated with axillary bud outgrowth. J. Exp. Bot. 2013, 64, 2307–2320, doi:10.1093/jxb/ert089.
[25]
Sun, F.; Guo, W.; Du, J.; Ni, Z.; Sun, Q.; Yao, Y. Widespread, abundant, and diverse TE-associated siRNAs in developing wheat grain. Gene 2013, 522, 1–7, doi:10.1016/j.gene.2013.03.101.
[26]
Zhao, Y.T.; Wang, M.; Fu, S.X.; Yang, W.C.; Qi, C.K.; Wang, X.J. Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes. Plant Physiol. 2012, 158, 813–823, doi:10.1104/pp.111.187666.
[27]
Henderson, I.R.; Zhang, X.Y.; Lu, C.; Johnson, L.; Meyers, B.C.; Green, P.J.; Jacobsen, S.E. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 2006, 38, 721–725, doi:10.1038/ng1804.
[28]
Daxinger, L.; Kanno, T.; Bucher, E.; van der Winden, J.; Naumann, U.; Matzke, A.J.; Matzke, M. A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation. EMBO J. 2009, 28, 48–57, doi:10.1038/emboj.2008.260.
[29]
Dunoyer, P.; Brosnan, C.A.; Schott, G.; Wang, Y.; Jay, F.; Alioua, A.; Himber, C.; Voinnet, O. An endogenous, systemic RNAi pathway in plants. EMBO J. 2010, 29, 1699–1712, doi:10.1038/emboj.2010.65.
[30]
Erhard, K.F.; Stonaker, J.L.; Parkinson, S.E.; Lim, J.P.; Hale, C.J.; Hollick, J.B. RNA polymerase IV functions in paramutation in zea mays. Science 2009, 323, 1201–1205, doi:10.1126/science.1164508.
[31]
Stonaker, J.L.; Lim, J.P.; Erhard, K.F., Jr.; Hollick, J.B. Diversity of Pol IV function is defined by mutations at the maize rmr7 locus. PLoS Genet. 2009, 5, e1000706, doi:10.1371/journal.pgen.1000706.
Wang, X.; Wang, H.; Wang, J.; Sun, R.; Wu, J.; Liu, S.; Bai, Y.; Mun, J.H.; Bancroft, I.; Cheng, F.; et al. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 2011, 43, 1035–1039, doi:10.1038/ng.919.
[34]
Stephenson, P.; Baker, D.; Girin, T.; Perez, A.; Amoah, S.; King, G.J.; Ostergaard, L. A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biol. 2010, 10, 62, doi:10.1186/1471-2229-10-62.
[35]
Cheng, F.; Liu, S.; Wu, J.; Fang, L.; Sun, S.; Liu, B.; Li, P.; Hua, W.; Wang, X. BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol. 2011, 11, p. 136. Available online: http://www.brassicadb.org/ (accessed on 1 August 2013).
[36]
Mosher, R.A.; Tan, E.H.; Shin, J.; Fischer, R.L.; Pikaard, C.S.; Baulcombe, D.C. An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs. PLoS One 2011, 6, e25756.
[37]
Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10, R25, doi:10.1186/gb-2009-10-3-r25.
[38]
Birchler, J.A.; Veitia, R.A. The gene balance hypothesis: From classical genetics to modern genomics. Plant Cell 2007, 19, 395–402, doi:10.1105/tpc.106.049338.
[39]
Haag, J.R.; Pikaard, C.S. Multisubunit RNA polymerases IV and V: Purveyors of non-coding RNA for plant gene silencing. Nat. Rev. Mol. Cell Biol. 2011, 12, 483–492, doi:10.1038/nrm3152.
[40]
Wierzbicki, A.T.; Cocklin, R.; Mayampurath, A.; Lister, R.; Rowley, M.J.; Gregory, B.D.; Ecker, J.R.; Tang, H.; Pikaard, C.S. Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Gene Dev. 2012, 26, 1825–1836, doi:10.1101/gad.197772.112.
[41]
Mi, S.J.; Cai, T.; Hu, Y.G.; Chen, Y.; Hodges, E.; Ni, F.R.; Wu, L.; Li, S.; Zhou, H.; Long, C.Z.; et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide. Cell 2008, 133, 116–127, doi:10.1016/j.cell.2008.02.034.
[42]
Havecker, E.R.; Wallbridge, L.M.; Hardcastle, T.J.; Bush, M.S.; Kelly, K.A.; Dunn, R.M.; Schwach, F.; Doonan, J.H.; Baulcombe, D.C. The arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 2010, 22, 321–334, doi:10.1105/tpc.109.072199.
[43]
Haag, J.R.; Pontes, O.; Pikaard, C.S. Metal A and metal B sites of nuclear RNA polymerases Pol IV and Pol V are required for siRNA-dependent DNA methylation and gene silencing. PLoS One 2009, 4, e4110.
[44]
Pereira, V. Insertion bias and purifying selection of retrotransposons in the Arabidopsis thaliana genome. Genome Biol. 2004, 5, R79, doi:10.1186/gb-2004-5-10-r79.
[45]
Lu, C.; Kulkarni, K.; Souret, F.F.; MuthuValliappan, R.; STej, S.S.; Poethig, R.S.; Henderson, I.R.; Jacobsen, S.E.; Wang, W.; Green, P.J.; et al. MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant. Genome Res. 2006, 16, 1276–1288, doi:10.1101/gr.5530106.
[46]
Mosher, R.A.; Melnyk, C.W.; Kelly, K.A.; Dunn, R.M.; Studholme, D.J.; Baulcombe, D.C. Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. Nature 2009, 460, 283–286, doi:10.1038/nature08084.
[47]
Huettel, B.; Kanno, T.; Daxinger, L.; Aufsatz, W.; Matzke, A.J.M.; Matzke, M. Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis. EMBO J. 2006, 25, 2828–2836, doi:10.1038/sj.emboj.7601150.
