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• W2146100687 abstract "Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences. Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences. MicroRNAs (miRNAs) are short, noncoding RNAs, about 21 nucleotides in length, with variable sequence complementarity to longer target RNAs. The typical target mRNA in animals has multiple motifs with limited sequence complementarity to one or more miRNAs in its 3′ untranslated region (UTR), and the predominant mode of miRNA action is translation inhibition or deadenylation-mediated mRNA degradation. In contrast, the typical target mRNA in plants has a single miRNA-complementary motif in the coding region with few or no mismatches, and the principal mode of action is transcript cleavage. Plant miRNAs have therefore often been compared to perfectly complementary short interfering RNAs (siRNAs) (Bartel, 2004Bartel D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (27026) Google Scholar, Filipowicz, 2005Filipowicz W. RNAi: The nuts and bolts of the RISC machine.Cell. 2005; 122: 17-20Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, Jones-Rhoades et al., 2006Jones-Rhoades M.W. Bartel D.P. Bartel B. MicroRNAs and their regulatory roles in plants.Annu. Rev. Plant Biol. 2006; 57: 19-53Crossref PubMed Scopus (1930) Google Scholar). The differences between animal and plant miRNAs are, however, not absolute. Human miR196 directs cleavage (Yekta et al., 2004Yekta S. Shih I.H. Bartel D.P. MicroRNA-directed cleavage of HOXB8 mRNA.Science. 2004; 304: 594-596Crossref PubMed Scopus (1347) Google Scholar), and at least one plant miRNA, miR172, acts both by preventing productive translation and by target cleavage (Aukerman and Sakai, 2003Aukerman M.J. Sakai H. Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes.Plant Cell. 2003; 15: 2730-2741Crossref PubMed Scopus (1372) Google Scholar, Chen, 2004Chen X. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.Science. 2004; 303: 2022-2025Crossref PubMed Scopus (1300) Google Scholar, Kasschau et al., 2003Kasschau K.D. Xie Z. Allen E. Llave C. Chapman E.J. Krizan K.A. Carrington J.C. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function.Dev. Cell. 2003; 4: 205-217Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, Lauter et al., 2005Lauter N. Kampani A. Carlson S. Goebel M. Moose S.P. MicroRNA172 down-regulates glossy15 to promote vegetative phase change in maize.Proc. Natl. Acad. Sci. USA. 2005; 102: 9412-9417Crossref PubMed Scopus (332) Google Scholar, Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar). MiRNAs are processed from larger precursors containing a self-complementary fold-back structure. Mature miRNAs are incorporated into RNA induced silencing complexes (RISCs) that include members of the ARGONAUTE (AGO) family of proteins. AGO proteins catalyze ribonucleolytic cleavage of the target at the position opposite of nucleotide 10 of the small RNA (Filipowicz, 2005Filipowicz W. RNAi: The nuts and bolts of the RISC machine.Cell. 2005; 122: 17-20Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, Kim, 2005Kim V.N. MicroRNA biogenesis: Coordinated cropping and dicing.Nat. Rev. Mol. Cell Biol. 2005; 6: 376-385Crossref PubMed Scopus (1872) Google Scholar). The 5′ portions of miRNAs and siRNAs are particularly important for guide function (Doench et al., 2003Doench J.G. Petersen C.P. Sharp P.A. siRNAs can function as miRNAs.Genes Dev. 2003; 17: 438-442Crossref PubMed Scopus (960) Google Scholar, Lewis et al., 2003Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (3949) Google Scholar, Mallory et al., 2004bMallory A.C. Reinhart B.J. Jones-Rhoades M.W. Tang G. Zamore P.D. Barton M.K. Bartel D.P. MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region.EMBO J. 2004; 23: 3356-3364Crossref PubMed Scopus (494) Google Scholar, Parizotto et al., 2004Parizotto E.A. Dunoyer P. Rahm N. Himber C. Voinnet O. In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA.Genes Dev. 2004; 18: 2237-2242Crossref PubMed Scopus (291) Google Scholar, Vaucheret et al., 2004Vaucheret H. Vazquez F. Crátá P. Bartel D.P. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development.Genes Dev. 2004; 18: 1187-1197Crossref PubMed Scopus (696) Google Scholar). In animals, part of the 5′ portion is called the “seed region,” and sequence complementarity to this region is often sufficient for target recognition (Brennecke et al., 2005Brennecke J. Stark A. Russell R.B. Cohen S.M. Principles of microRNA-target recognition.PLoS Biol. 2005; 3: e85Crossref PubMed Scopus (1748) Google Scholar, Lewis et al., 2005Lewis B.P. Burge C.B. Bartel D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9265) Google Scholar). The importance of the 5′ region applies also to plant miRNAs. For example, introduction of mismatches between positions 3 and 11 drastically reduces miRNA-guided cleavage of PHABULOSA (PHB) mRNA by miR165, with mutations in the 3′ region having much weaker effects (Mallory et al., 2004bMallory A.C. Reinhart B.J. Jones-Rhoades M.W. Tang G. Zamore P.D. Barton M.K. Bartel D.P. MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region.EMBO J. 2004; 23: 3356-3364Crossref PubMed Scopus (494) Google Scholar). These observations are consistent with global rules of miRNA target interaction that have been experimentally deduced from transcript profiling of plants overexpressing natural or artificial miRNAs (Schwab et al., 2006Schwab R. Ossowski S. Riester M. Warthmann N. Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis.Plant Cell. 2006; 18: 1121-1133Crossref PubMed Scopus (953) Google Scholar, Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar). Many plant miRNAs are encoded by gene families, which can have more than a dozen members. Some miRNAs originating from different family members are identical, while others differ in up to three nucleotides, typically near the 3′ end of the mature miRNA. Whether these differences reflect functional specialization of miRNA family members is not known. The Arabidopsis genome contains several genes that can potentially produce five distinct miRNAs belonging to the miR159 and miR319 families, which share 17 identical nucleotides but have 5′ ends that are offset by a single nucleotide (Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar, Reinhart et al., 2002Reinhart B.J. Weinstein E.G. Rhoades M.W. Bartel B. Bartel D.P. MicroRNAs in plants.Genes Dev. 2002; 16: 1616-1626Crossref PubMed Scopus (1585) Google Scholar, Sunkar and Zhu, 2004Sunkar R. Zhu J.K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis.Plant Cell. 2004; 16: 2001-2019Crossref PubMed Scopus (1432) Google Scholar). The effects of overexpression, as well as the position of target cleavage, have indicated that miR159a and miR319a (also known as miR-JAW) have largely nonoverlapping effects in vivo. MiR159 targets several MYB transcription factor genes involved in flowering and male fertility, while miR319 primarily affects a clade of TCP transcription factor genes controlling leaf shape (Achard et al., 2004Achard P. Herr A. Baulcombe D.C. Harberd N.P. Modulation of floral development by a gibberellin-regulated microRNA.Development. 2004; 131: 3357-3365Crossref PubMed Scopus (592) Google Scholar, Millar and Gubler, 2005Millar A.A. Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.Plant Cell. 2005; 17: 705-721Crossref PubMed Scopus (453) Google Scholar, Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar, Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar). We have analyzed the basis for the different biological effects of miR159 and miR319, and we show with several approaches that both differences in sequence and expression contribute to specific interactions between these two miRNAs and their targets. Mobility of miR319a/b during gel electrophoresis, as well as cloning of miR319c, had suggested that these miRNAs are predominantly 20 nucleotides in length (Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar, Sunkar and Zhu, 2004Sunkar R. Zhu J.K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis.Plant Cell. 2004; 16: 2001-2019Crossref PubMed Scopus (1432) Google Scholar). More recent deep sequencing efforts have shown that, as with other miRNAs, there is some heterogeneity in size, but the 21-nucleotide-long variants appear to dominate (Table 1; http://asrp.cgrb.oregonstate.edu/) (Fahlgren et al., 2007Fahlgren N. Howell M.D. Kasschau K.D. Chapman E.J. Sullivan C.M. Cumbie J.S. Givan S.A. Law T.F. Grant S.R. Dangl J.L. Carrington J.C. High-throughput sequencing of Arabidopsis microRNAs: Evidence for frequent birth and death of MIRNA genes.PLoS ONE. 2007; 2: e219Crossref PubMed Scopus (872) Google Scholar, Rajagopalan et al., 2006Rajagopalan R. Vaucheret H. Trejo J. Bartel D.P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.Genes Dev. 2006; 20: 3407-3425Crossref PubMed Scopus (998) Google Scholar). MiR159 and miR319 can potentially be generated from six different precursors. All of the mature miRNAs have been identified in small RNA libraries, although at very different levels (Table 1). The fold-back structures of miR159 and miR319 precursors have similar lengths, and both show sequence conservation in other species outside of the miRNA/miRNA∗ base-paired region. In addition, the miRNAs all arise from an equivalent position within the 3′ arm of the fold-back (see Figure S1 in the Supplemental Data available with this article online). While precursors from both families are more similar to each other than the average random pair of miRNA precursors, both by sequence and structural criteria (Figure S2), their similarity is not exceptionally high. Thus, though they are often treated as a single family (Jones-Rhoades and Bartel, 2004Jones-Rhoades M.W. Bartel D.P. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA.Mol. Cell. 2004; 14: 787-799Abstract Full Text Full Text PDF PubMed Scopus (1699) Google Scholar), it remains unclear whether miR159 and miR319 originated from a common ancestor.Table 1miR159/miR319 FamiliesmiRNASequenceTimes Clonedahttp://asrp.cgrb.oregonstate.edu/; total numbers from all genotypes and tissues; small RNAs unambiguously assigned to wild-type given in parentheses.Overexpression PhenotypebAt least 80 T1 plants per construct.Cotyledon EpinastyCrinkled LeavesStamen DefectsmiR159a.uUUGGAuUGAAGGGAGCUCua19,940 (4,306)--∼48%miR159b.uUUGGAuUGAAGGGAGCUCuu1,982 (387)--∼46%miR159c.uUUGGAuUGAAGGGAGCUCcu21 (3)---miR319a..UUGGAcUGAAGGGAGCUCcc/..UUGGAcUGAAGGGAGCUCccu6 (0)/145 (45)Severe (>90%)Strong (>90% of T1)∼33%miR319bSevere (>90%)Strong (>80% of T1)Not determinedmiR319c.uUUGGAcUGAAGGGAGCUCcu/..UUGGAcUGAAGGGAGCUCcuu12 (3)/17 (8)Moderate (>50%)Mostly Normal∼24%a http://asrp.cgrb.oregonstate.edu/; total numbers from all genotypes and tissues; small RNAs unambiguously assigned to wild-type given in parentheses.b At least 80 T1 plants per construct. Open table in a new tab Two groups of miR159/miR319 targets have previously been experimentally validated (Figures 1A and 1B). These include several members of the GAMYB-related clade of MYB transcription factor genes, which have important roles in hormone response and male fertility, and five TCP genes related to CINCINNATA from snapdragon, which control leaf growth (Achard et al., 2004Achard P. Herr A. Baulcombe D.C. Harberd N.P. Modulation of floral development by a gibberellin-regulated microRNA.Development. 2004; 131: 3357-3365Crossref PubMed Scopus (592) Google Scholar, Millar and Gubler, 2005Millar A.A. Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.Plant Cell. 2005; 17: 705-721Crossref PubMed Scopus (453) Google Scholar, Nath et al., 2003Nath U. Crawford B.C. Carpenter R. Coen E. Genetic control of surface curvature.Science. 2003; 299: 1404-1407Crossref PubMed Scopus (523) Google Scholar, Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar, Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar). A third group of targets comprises the recently described DUO1/MYB125 gene, which has an essential function in the male germline and which defines a unique MYB clade in Arabidopsis (Rotman et al., 2005Rotman N. Durbarry A. Wardle A. Yang W.C. Chaboud A. Faure J.E. Berger F. Twell D. A novel class of MYB factors controls sperm-cell formation in plants.Curr. Biol. 2005; 15: 244-248Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). We confirmed that mRNAs encoding DUO1 as well as MYB101, a member of the GAMYB clade, are cleaved in wild-type plants in a position that indicates targeting by miR159 (Figure 1C) (Reyes and Chua, 2007Reyes J.L. Chua N.H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination.Plant J. 2007; 49: 592-606Crossref PubMed Scopus (533) Google Scholar). MiR159 regulation of DUO1 is likely important for its normal function, as shown by overexpression of a form with synonymous mutations that disrupt the miRNA target site (Figure S3). Computational predictions using experimentally established criteria (Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar) suggest that miR319 can target five TCP genes but also several MYB genes. In contrast, miR159a and miR159b should be specific for MYB genes (Figure 1D). Only miR159c would be predicted to target two of five TCP genes, but its expression levels are two to three orders of magnitude lower than those of miR159a and miR159b. In an alignment, the 5′ ends of miR159a and miR159b are offset by one nucleotide relative to miR319a/b. Because AGO catalyzes cleavage of targets invariably opposite of the bond between nucleotides 10 and 11 from the 5′ end of the miRNA, cleavage products will differ by one nucleotide depending on which miRNA guides the processing event (Figure 1E). Cleavage site mapping and miRNA overexpression have indicated that the MYB genes are predominantly targeted by miR159 in vivo, while TCP genes are targeted by miR319a/b (Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar, Schwab et al., 2005Schwab R. Palatnik J.F. Riester M. Schommer C. Schmid M. Weigel D. Specific effects of microRNAs on the plant transcriptome.Dev. Cell. 2005; 8: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar) (Figure 1E). Overexpression of the endogenous MIR319a gene in jaw-D mutants leads to epinastic cotyledons and crinkly leaves (Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar). We found that plants overexpressing miR319a from the cauliflower mosaic virus 35S promoter had cotyledon and leaf phenotypes similar to those of jaw-D mutants, but some 35S:miR319a plants had additional stamen defects resembling those of 35S:miR159a plants, which are male sterile due to inactivation of MYB33 and MYB65 (Achard et al., 2004Achard P. Herr A. Baulcombe D.C. Harberd N.P. Modulation of floral development by a gibberellin-regulated microRNA.Development. 2004; 131: 3357-3365Crossref PubMed Scopus (592) Google Scholar, Millar and Gubler, 2005Millar A.A. Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.Plant Cell. 2005; 17: 705-721Crossref PubMed Scopus (453) Google Scholar) (Figures 2A and 2B). They also had stronger leaf defects than jaw-D mutants, suggesting that the stamen defects were due to particularly high levels of miR319a in these plants (Figure 2B). Although the majority of 35S:miR319c plants had only weakly epinastic cotyledons and relatively normal leaves, many had stamen defects (Figure 2A). MiR319c accumulated to moderate levels in these plants (Figure 2C), and it is possible that higher levels may have resulted in crinkled leaves as well. Thus, strong overexpression of miR319a or moderate overexpression of miR319c causes a male sterility phenotype similar to that seen in miR159 overexpressers. We previously analyzed 22 MYB33 and MYB65 cleavage products and found that the 5′ end of only one MYB65 product was compatible with cleavage guided by miR319a/b (Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar). To determine whether this constitutes a real but rare event, we developed a rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) assay to distinguish between miR159 and miR319-triggered cleavage products (for details, see Figure S4). This assay confirmed that MYB33, which has a target site identical to that of MYB65, is indeed occasionally targeted by miR319a in wild-type plants (Figure 3A). MiR319-triggered cleavage products of MYB33 are more easily detected in strong 35S:miR319a overexpressers (Figure S4). A transgene reporting expression of the MIR319a primary transcript revealed transient expression in young floral buds (Figure S5), in which the MYB33 promoter is active as well (Millar and Gubler, 2005Millar A.A. Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.Plant Cell. 2005; 17: 705-721Crossref PubMed Scopus (453) Google Scholar). Thus, there is at least some overlap of miR319 and MYB33 expression, although miR159 is expressed much more widely and more strongly than miR319. Furthermore, the MYB33 and MYB65 promoters are strongly active in many tissues and stages in which we could not detect MIR319a promoter activity, thus indicating that both differences in expression level and pattern contribute to the preferred targeting of MYB mRNAs by miR159. The rare cross-regulation of MYB targets by miR319a/b raised the question of whether it is simply impossible to generate a TCP-specific miRNA or whether cross-regulation is tolerated by the plant as an infrequent and therefore largely innocuous event. Using the Web MicroRNA Designer (WMD) tool (Schwab et al., 2006Schwab R. Ossowski S. Riester M. Warthmann N. Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis.Plant Cell. 2006; 18: 1121-1133Crossref PubMed Scopus (953) Google Scholar), we designed two miR319a variants that should retain their activity toward the TCPs at the expense of interaction with the MYBs. Changes at positions 14, 15, and 17 were sufficient to strongly reduce the ability of miR319 to cause male sterility, indicative of reduced MYB targeting, while retaining the ability to induce altered leaf morphology, indicative of normal TCP targeting (Figure 3C). Additional modification of positions 19 and 20 did not further modify miR319a overexpression phenotype (Figure 3D). In contrast to cross-regulation of MYB genes by miR319a, computational predictions, cleavage site mapping, and overexpression phenotypes suggest that despite their high expression levels, miR159a and miR159b have no effect on TCP genes (Achard et al., 2004Achard P. Herr A. Baulcombe D.C. Harberd N.P. Modulation of floral development by a gibberellin-regulated microRNA.Development. 2004; 131: 3357-3365Crossref PubMed Scopus (592) Google Scholar, Millar and Gubler, 2005Millar A.A. Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.Plant Cell. 2005; 17: 705-721Crossref PubMed Scopus (453) Google Scholar, Palatnik et al., 2003Palatnik J.F. Allen E. Wu X. Schommer C. Schwab R. Carrington J.C. Weigel D. Control of leaf morphogenesis by microRNAs.Nature. 2003; 425: 257-263Crossref PubMed Scopus (1293) Google Scholar) (Figure 1). We were unable to obtain plants overexpressing miR159c, which can potentially target two TCP genes (Figure 1D), although we detected expression of the MIR159c precursor in 35S:miR159c transgenic lines (Figure S6). In addition, we did not detect an increase in miR159c levels when the 35S:miR159c construct was transiently introduced into N. benthamiana leaves (Figure 4A). It therefore appears that the low miR159c levels in normal plants are at least partially due to inefficient processing. Together, these findings suggest that the potential effect of miR159c on TCPs is not relevant in vivo, consistent with the observation that we have never seen a cleavage product indicative of TCP targeting by miR159c. A shortcoming of the evidence presented so far is that we could not exclude translational effects of miR159a/b on TCPs, similar to the translational inhibition caused by miRNAs, with limited sequence complementarity to their targets in animals. To analyze translational repression by miRNAs, we transiently expressed a TCP4:GFP fusion protein in N. benthamiana leaves using A. tumefaciens. As expected, coexpression of miR319a led to strong reduction of TCP4:GFP transcript levels and GFP fluorescence (Figures 4A and 4B). Even the weaker expression of miR319b (which is identical to miR319a) from a 35S:miR319b construct had a pronounced effect on TCP4:GFP RNA levels (Figure 4B). As a control, we prepared a version of TCP4:GFP with multiple mutations in the miRNA complementary motif. MiR319 coexpression had no effect on mTCP4:GFP RNA or protein (Figures 4A and 4B). We further tested susceptibility of TCP4:GFP mRNA to miR319a in a semiquantitative fashion. Even after coinfiltration with a 100-fold dilution of the 35S:miR319a vector, we detected some degradation of TCP4:GFP mRNA, indicating exquisite sensitivity of TCP4 to miR319-guided cleavage (Figure S7). Although endogenous levels of miR159 are substantial in N. benthamiana leaves (Figure 4A), TCP4:GFP mRNA and fluorescence are easily detected when N. benthamiana leaves are not coinfiltrated with a 35S:miR319 construct, indicating that miR159 affects neither TCP4:GFP mRNA nor protein. We attempted to further increase miR159 levels by coinfiltrating 35S:miR159a or 35S:miR159b constructs, but no obvious effects on mRNA levels or GFP fluorescence were seen (Figure 4B). These findings are in agreement with the observation that miR159 overexpression in Arabidopsis does not cause epinastic cotyledons or crinkly leaves, which would be the consequences of TCP inactivation. All our experiments indicated that miR159 specificity is encoded in the miRNA itself. Further support for this comes from 1H nuclear magnetic resonance (NMR) spectroscopy, which revealed differences in the in vitro structures of miR159-MYB33 and miR159-TCP4 RNA duplices (Figure S8). To identify nucleotide positions that underlie the differential interaction of miR159a with MYB and TCP mRNAs in vivo, we turned to site-directed mutagenesis. Mutant miRNA variants were expressed in the context of a genomic MIR159a fragment, which had no effects on leaf morphogenesis in its wild-type form (Figure 5A). As a control, we expressed the MIR319a fold-back under control of MIR159a genomic sequences. The strongly crinkled leaves and epinastic cotyledons caused by this transgene (Figure 5G), similar to those seen in 35S:miR319a plants, indicated that the much higher levels of miR159a in plants compared to those of miR319 are at least partially due to broader and more extensive activity of MIR159a regulatory sequences. The results also confirmed that despite their overlapping expression, TCPs are normally not targeted by miR159. At a difference with 35S:miR319a plants, the expression of miR319a from the MIR159a regulatory sequences did not induce male sterility, which is consistent with limited expression of miR159 in tissues where MYB activity is required. Thus, miR319a-like activity of miR159a variants should be detectable without ectopic expression. Nevertheless, we tested the mutant precursors also under the control of the 35S promoter to account for potentially lower expression of the mutant miRNAs. As for the miR319a variants, compensatory mutations were introduced in the miR159a∗ sequence to maintain the secondary structure of the precursor (Figure S9). All of the mutant miRNAs accumulated in transgenic plants, as shown with small RNA blots, although there were differences in signal intensity. While some of this might be due to variation in miRNA levels, experiments with synthetic miRNAs showed that mutant miRNAs also hybridize less efficiently to the standard miR159a probe (Figure 5H and Figure S10). In addition, some of the mutant miRNAs migrated differently from endogenous miR159a, which is probably caused by distinct secondary structures of the miRNAs (Figure S10). We introduced point mutations to make miR159a successively more similar to miR319a. Converting the G:U pair at position 7 to a perfect match did not enable miR159a to affect leaf morphogenesis through TCP targeting (Figure 5B). That a match at this position alone is neither essential nor sufficient for miRNA activity can also be inferred from validated miR159a targets, such as MYB33 and MYB65, which contain a complete mismatch at position 7 (Figure 1E). We next mutated the 3′ end of the miR159a sequence, eliminating two mismatches to TCP4 (because of a different sequence at the extreme 3′ end, this would correspond only to a single change in the case of miR159b). Again, the resulting miRNA triggered no miR319a-like effects on leaf morphology (Figure 5C). However, when the 5′ and 3′ mutations were combined in a doubly mutant miRNA, leaf morphology defects typical for miR319a were seen (Figure 5D). Ex" @default.
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• W2146100687 date "2007-07-01" @default.
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• W2146100687 title "Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319" @default.
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