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. 2016 Jul 6;172(1):297–312. doi: 10.1104/pp.16.00830

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels1[OPEN]

Jakub Dolata 1,2, Mateusz Bajczyk 1,2, Dawid Bielewicz 1,2, Katarzyna Niedojadlo 1,2, Janusz Niedojadlo 1,2, Halina Pietrykowska 1,2, Weronika Walczak 1,2, Zofia Szweykowska-Kulinska 1,2,*, Artur Jarmolowski 1,2,*
PMCID: PMC5074614  PMID: 27385819

Arabidopsis ARGONAUTE 1, in addition to its well-known role in mRNA target cleavage and miRNA-mediated translation inhibition, is involved in the cotranscriptional regulation of MIR gene expression.

Abstract

Plants as sessile organisms have developed prompt response mechanisms to react to rapid environmental changes. In addition to the transcriptional regulation of gene expression, microRNAs (miRNAs) are key posttranscriptional regulators of the plant stress response. We show here that the expression levels of many miRNAs were regulated under salt stress conditions. This regulation occurred at the transcriptional and posttranscriptional levels. During salinity stress, the levels of miRNA161 and miRNA173 increased, while the expression of pri-miRNA161 and pri-miRNA173 was down-regulated. Under salt stress conditions, miRNA161 and miRNA173 were stabilized in the cytoplasm, and the expressions of MIR161 and MIR173 were negatively regulated in the nucleus. ARGONAUTE1 (AGO1) participated in both processes. We demonstrated that AGO1 cotranscriptionally controlled the expression of MIR161 and MIR173 in the nucleus. Our results suggests that AGO1 interacts with chromatin at MIR161 and MIR173 loci and causes the disassembly of the transcriptional complex, releasing short and unpolyadenylated transcripts.

MicroRNAs (miRNAs) are short (21–24 nt) RNA molecules that control gene expression at the posttranscriptional level by cleavage of mRNA targets or by inhibition of their translation (Llave et al., 2002; Reinhart and Bartel, 2002; Palatnik et al., 2003; Li et al., 2013). miRNA biogenesis is a multistep process that involves various proteins, including a member of the RNase III type endoribonuclease family, which is responsible for the enzymatic activity in the miRNA processing complex. In contrast to animal cells, plant miRNA biogenesis occurs entirely in the cell nucleus. All plant miRNA genes (MIRs) are transcribed exclusively by RNA polymerase II (RNAPII), and long primary MIR transcripts (pri-miRNAs) must be precisely processed to produce mature and active miRNAs. The 5′ end of the miRNA primary precursor, similar to all other RNAPII transcripts, is protected by a specific cap structure that is recognized and bound by the nuclear cap-binding protein complex (CBC) consisting of two subunits: CBP20 and CBP80 (Hugouvieux et al., 2001; Kmieciak et al., 2002; Daszkowska-Golec et al., 2013). The 3′ end of pri-mRNA is modified by the cleavage and polyadenylation machinery that adds a poly(A) tail to the 3′ end of all pri-mRNAs. Moreover, many plant pri-miRNAs, like most mRNA precursors (pre-mRNA), contain introns that are excised by the spliceosome (Szarzynska et al., 2009; Kruszka et al., 2013; Zielezinski et al., 2015). We have recently shown that the splicing of such introns from pri-miRNAs, or more precisely their active 5′ splice sites, stimulates miRNA maturation (Bielewicz et al., 2013; Schwab et al., 2013).

The most characteristic feature of pri-miRNA is an imperfect stem-loop structure in which a miRNA sequence is embedded. Plant pri-miRNAs are processed in a two-step process that is catalyzed by a single endoribonuclease known as DICER-LIKE1 (DCL1). To achieve a high efficiency and precision of cutting, the DCL1 endoribonuclease must cooperate with at least two other proteins: HYPONASTIC LEAVES1 (HYL1; Vazquez et al., 2004; Park et al., 2002; Han et al., 2004), which is a dsRNA binding protein, and a zinc-finger-containing protein called SERRATE (SE; Dong et al., 2008; Laubinger et al., 2008; Raczynska et al., 2014). During the first step of plant miRNA biogenesis, a hairpin structure (pre-miRNA) is generated from a long primary miRNA precursor, whereas during the second step, a miRNA/miRNA* duplex is released from the pre-miRNA. Both of these cleavages are catalyzed by DCL1 (Tang et al., 2003; Kurihara and Watanabe, 2004). Next, miRNA-containing duplexes are methylated by the HUA ENHANCER1 methylase, and the duplexes are then exported to the cytoplasm via the HASTY-mediated export pathway (Park et al., 2005). In the cytoplasm, the miRNA strand of each duplex interacts with the ARGONAUTE1 (AGO1) protein, which is an essential part of the RNA-induced silencing complex (RISC) that is directly involved in the regulation of gene expression. In addition to DCL1, HYL1, and SE, which combine with a miRNA precursor to form the core of the plant microprocessor, many other proteins have been ascribed to miRNA biogenesis in plants, e.g. DAWDLE, which participates in pri-miRNA recognition and enhances access of pri-miRNA for DCL1 (Yu et al., 2008), MOS2, which promotes pri-miRNA processing (Wu et al., 2013), and C-TERMINAL DOMAIN PHOSPHATASE LIKE1 (CPL1), which is crucial for HYL1 dephosphorylation and consequently for proper pri-miRNA processing and miRNA strand selection (Manavella et al., 2012).

Because miRNAs play crucial roles in the control of plant growth and development, the miRNA biogenesis pathway must be tightly controlled. This regulation of miRNA expression can be achieved at several levels. It also includes the control of the level of DCL1 and AGO1 mRNA by specific miRNAs (Meng et al., 2011) in a distinctive feedback mechanism. The final accumulation of miRNA molecules and consequently the final levels of their mRNA targets result from a combined effect of the regulation of MIR genes transcription as well as the processing of miRNA precursors (Kai and Pasquinelli, 2010; Barciszewska-Pacak et al., 2015). The final levels of mature miRNAs can also be regulated by miRNA degradation or stabilization (Meng et al., 2011). The 10-member Arabidopsis (Arabidopsis thaliana) family of ARGONAUTE proteins is involved in such miRNA stabilization (Kai and Pasquinelli, 2010). It has been demonstrated that miR168 is stabilized by AGO1 (Vaucheret et al., 2006; Vaucheret, 2009). The main role of Arabidopsis AGO1 as a slicer in the RISC complex that cuts an mRNA target places AGO1 in the cytoplasm where the cleavage of miRNA targets occurs, yet there are experimental data suggesting that AGO1 can also be localized in the nucleus in plant cells (Ye et al., 2012; Duan et al., 2012). However, the role of AGO1 in this cell compartment remains elusive. Interestingly, in human cells, one member of the AGO family, AGO1, regulates gene expression directly by influencing RNAPII (Huang et al., 2013), and AGO1 and AGO2 both affect pre-mRNA splicing by modulating the processivity of RNAPII (Ameyar-Zazoua et al., 2012).

It is well known that stress conditions strongly influence miRNA biogenesis by enhancing or repressing the transcription of specific MIR genes and by affecting the activities of the proteins responsible for miRNA maturation. Consequently, this process leads to changes in the expression levels of specific miRNA targets. Particularly in plants, which are sessile organisms, such rapid and effective responses to different environmental stimuli are crucial for the proper acclimatization to new conditions. Many miRNAs have been ascribed to different biotic and abiotic stresses (Kruszka et al., 2012; Barciszewska-Pacak et al., 2015); however, only in very few examples is the mechanism underlying the regulation of such miRNA expression understood. Therefore, to understand the mechanisms underlying the regulation of miRNA expression in plants, we investigated the influence of salinity stress on the miRNA Arabidopsis transcriptome by applying the qPCR-based platform mirEX that was recently developed in our laboratory and allows us to monitor the levels of all known Arabidopsis pri-miRNAs (Bielewicz et al., 2012; Zielezinski et al., 2015). We identified miRNAs with general expression levels that changed in response to salt stress during transcriptional and/or posttranscriptional steps of their biogenesis. Here, we show that, in addition to its well-described role in mRNA cleavage, plant AGO1 is also involved in the cotranscriptional regulation of specific miRNAs that are responsive to salt stress.

RESULTS

Salt Stress Induces Changes in the Expression of Many Arabidopsis MIR Genes

To evaluate the influence of salt stress on plant miRNA biogenesis, we used an in vitro cell suspension system. The widely used T87 cell line, which originated from Arabidopsis Col-0 leaves, is very easily induced by different abiotic stresses (Yamada et al., 2004; Sokol et al., 2007; Sasaki et al., 2008; Li et al., 2012a; Ludwikow et al., 2014). In addition, T87 cell culture permits the observation of molecular responses to stress conditions in homogenous cell populations, facilitating the analysis and interpretation of results. Recently, we developed a high-throughput real-time quantitative PCR (RT-qPCR)-based platform, mirEX, which can be applied for the simultaneous examination of the accumulation of all known Arabidopsis miRNA precursors (pri-miRNAs). Our mirEX platform has been demonstrated to be a useful tool for the global profiling of miRNA expression during different developmental stages, mutants, and/or Arabidopsis plants growing under various environmental conditions (Zielezinski et al., 2015; Barciszewska-Pacak et al., 2015). We applied the mirEX panel to investigate the influence of salt stress on MIR gene expression in T87 cells. First, we performed experiments to establish a proper concentration of NaCl for use in our salinity tests. Moreover, the best time to treat T87 cells with salt was also estimated in these test experiments. The results showed that T87 cells survive in the presence of 250 mm NaCl (Supplemental Fig. S1), and the highest level of the stress response (monitored by the highest expression level of well-characterized salinity stress gene markers; Kreps et al., 2002) was obtained after 60 min of treatment of the cells with 250 mm NaCl (Supplemental Fig. S2). Therefore, we stressed the T87 cells with 250 mm NaCl for 60 min for our studies.

The data collected from the experiments that were performed under the established stress conditions clearly indicate that salt stress greatly influences the expression pattern of Arabidopsis MIR genes: 40% (116 of 289) of all pri-miRNAs were significantly changed after NaCl treatment of T87 cells. Most of these pri-miRNAs (63; 22%) were down-regulated compared with the control untreated cells. However, there was also a large group of up-regulated miRNA precursors (53; 18%) (Fig. 1; Supplemental Table S1). We also assessed the general expression level of MIRs that were affected by salt stress in cells growing under nonstress conditions. Interestingly, most of the up-regulated pri-miRNAs during salt stress were expressed at low levels under standard conditions of T87 cell growth, and the miRNA precursors that were down-regulated after salt treatment were highly or moderately expressed under nonstress conditions (Supplemental Fig. S3).

Figure 1.

Figure 1.

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Changes in the accumulation of Arabidopsis miRNA precursors under salt stress monitored by RT-qPCR. n = 3; t test P value ≤ 0.05.

miRNA161 and miRNA173 Levels Are Increased in Response to Salt Stress

To obtain information regarding the effects of salt on the production and accumulation of mature Arabidopsis miRNAs, we examined the expression levels of selected miRNAs before and after salt treatment by northern-blot analysis. In this experiment, we assessed the levels of the miRNAs that originated from the precursors whose accumulation levels were modified under salinity stress conditions. In eight cases, the increased levels of the analyzed MIR transcripts correlated with higher amounts of mature miRNAs, and vice versa, the lower pri-miRNA levels correlated with miRNAs that were also down-regulated. We concluded, therefore, that this group of salt-responsive MIR genes was most likely regulated at the transcriptional level in response to salt stress (Fig. 2A; Supplemental Fig. S4A). We also noticed nine examples in which the final mature miRNA level resulted from differentially expressed MIR genes from the same family (e.g. miRNA169, miRNA390, and miRNA395 families; Supplemental Fig. S4B), indicating a precise and gene-specific regulation of the levels of these miRNAs. However, in addition to the examples of the transcriptional regulation of MIR gene expression in response to salinity stress, we observed seven MIRs that showed different responses in the levels of the pri-miRNA and its cognate miRNAs after NaCl treatment. Two such examples in which the level of the pri-miRNA decreased, while the level of the mature miRNAs increased, were miRNA161 and miRNA173 (Fig. 2B; other examples of miRNAs that do not follow a positive correlation in pri-miRNA and miRNA levels are presented in Supplemental Fig. S4C). MIR403 was selected as an example of a miRNA gene that did not respond to salt stress (Supplemental Fig. S4D).

Figure 2.

Figure 2.

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The levels of selected pri-miRNAs and miRNAs in Arabidopsis T87 cells in response to salinity stress. RT-qPCR (pri-miRNAs) and northern hybridization (miRNAs) of A, salt stress-responsive miRNAs showing a positive correlation between the mature miRNA and its precursor, and B, salt stress-responsive miRNAs showing a negative correlation between the mature miRNA and its precursor. The values presented in the charts are the mean ± sd (n = 3) fold change between stress and control conditions. The northern hybridization values were normalized to the U6 level and are presented as the percentage change in comparison to the control conditions (100%).

To ensure that our observations regarding the higher levels of miRNA161 and miRNA173 in response to salinity stress indicated fully active miRNA161 and miRNA173 molecules, we also examined the levels of the mRNA targets of these two miRNAs before and after salt treatment (Fig. 3A). In parallel, we analyzed the levels of the mRNA targets of selected miRNAs that are transcriptionally regulated in response to salt stress: miRNA163, miRNA165, and miRNA828 (Fig. 3B). In all cases, an expected negative correlation between the levels of miRNA and their mRNA targets was observed (Fig. 3). Because miRNA 173 targets TAS1 and TAS2 transcripts that after miRNA-directed cleavage are processed into ta-siRNAs, the level of selected ta-siRNAs from TAS1 and TAS2 were also tested. As expected, the levels of these ta-siRNAs increased after salt treatment (Supplemental Fig. S5D). This correlates with the elevated level of miRNA173 (Fig. 2B) and the decreased levels of TAS1 and TAS2 transcripts (Fig. 3A).

Figure 3.

Figure 3.

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Functionality of salt-responsive miRNAs. RT-qPCR analyses of target mRNAs in T87 cells under normal and stress conditions. A, The levels of the miRNA161 and miRNA173 targets. B, The levels of the miRNA163, miRNA165, and miRNA828 targets. The values presented in the charts are the mean ± sd (n = 3) fold change between stress and control conditions.

The results led us to select miRNA161 and miRNA173 as NaCl-responsive miRNAs that are regulated not at the transcriptional level but most likely at the posttranscriptional level, are functional and are able to induce the cleavage of specific mRNA targets.

miRNA161 and miRNA173 Are Responsive to Salt Stress Both in T87 Cell Suspensions and 2-Week-Old Arabidopsis Seedlings

As previously mentioned, in salt-stressed T87 cells, we observed a negative correlation between the accumulation of pri-miRNA161 and pri-miRNA173 and the levels of mature miRNA161 and miRNA173, respectively (Fig. 2B). To ensure that this result was not limited to the cell culture conditions, we performed similar salt stress experiments in parallel utilizing two-week-old Arabidopsis Col-0 seedlings. The conditions applied in the seedling stress experiment were carefully established using well-known gene markers of salinity to ensure the systems were fully comparable (Supplemental Fig. S6). In the seedlings, we tested the levels of pri-miRNAs and miRNAs that were previously identified as salt-responsive in the T87 cell system. Salinity stress was induced by 250 mm NaCl, and the levels of pri-miRNAs and miRNAs were monitored by RT-qPCR and northern-blot analysis, respectively, after 120, 180, and 360 min of salt treatment. Similar results were obtained for all tested miRNAs in both experimental systems (Supplemental Fig. S7). In experiments conducted in Arabidopsis seedlings, we applied 250 mm NaCl for 120 min. These conditions were fully comparable to the conditions used in the T87 cell system (250 mm NaCl for 60 min) in terms of the miRNA response to salt stress.

The seedling experiments clearly supported the salt-responsive characteristics of miRNA161 and miRNA173 in T87 cells and in planta (Supplemental Fig. S7B). Moreover, in both experimental systems, a negative correlation between pri-miRNAs and their mature cognate miRNAs was observed. The levels of pri-miR403 and miRNA403 were not affected by salt treatment, as observed in experiments in which T87 cells were used. Therefore, MIR403 was selected as a control for our further experiments because it was not modulated in response to salt stress at either the level of the precursor or the mature miRNA molecule (Supplemental Figs. S4D and S7C).

AGO1 Stabilizes miRNA161 and miRNA173 during Salinity Stress

It is known that single-stranded RNA molecules are degraded in animal cells by XRN-2 (a 5′-3′ exonuclease), and miRNAs are usually protected if they are incorporated into Argonaute-containing complexes (Chatterjee and Grosshans, 2009; Winter and Diederichs, 2011). We hypothesized that the accumulation of miRNA161 and miRNA173 during salt stress could be due to the stabilization of both miRNA molecules in AGO1-containing complexes. If our hypothesis regarding miRNA161 and miRNA173 stabilization during salt stress is correct, the accumulation effect should be limited to the acting miRNA molecules but not their passenger strands, miRNA161* and miRNA173*, according to the “use it or lose it” rule (Kai and Pasquinelli, 2010). The northern hybridization assay revealed that, indeed, miRNA161* and miRNA173* behaved differently than their partner miRNAs. Neither of the tested miRNAs* accumulated in response to salt stress, although the levels of miRNA161 and miRNA173 increased under the same experimental conditions (Fig. 4A).

Figure 4.

Figure 4.

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Stabilization of miRNA161 and miRNA173 by AGO1 under salt stress conditions. A, Northern-blot analyses of miRNA161* and miRNA173* in response to salt stress. B, The enrichment of miRNA161 and miRNA173 in AGO1-containing complexes (RNA IP during salinity stress). The data are presented as the mean ± sd (n = 3) % of input during stress and control conditions. C, The fold change of miRNA161 and miRNA173 in wild type (Ler) and the ago1-11 mutant in stress conditions. The values obtained by northern hybridization are presented in the chart as the mean ± sd (n = 3) fold change between stress and control conditions. Values were normalized to the U6 level; * t test P value ≤ 0.05.

AGO1 homeostasis in Arabidopsis results from the interplay between the transcriptional regulation of the AGO1 gene and the posttranscriptional repression of its expression by miRNA168. It has been shown that AGO1 protein levels in plants increase during many different stresses, including salt stress (Vaucheret et al., 2006; Vaucheret 2009; Várallyay et al., 2010; Li et al., 2012b). To investigate whether a similar accumulation of AGO1 could also be observed under the experimental conditions used in our studies, we examined changes in AGO1 expression in Arabidopsis seedlings treated with NaCl at the transcript and protein levels. As expected, AGO1 mRNA and AGO1 protein both accumulated in response to the salt stress applied in our experiments (Supplemental Fig. S8).

To confirm that the AGO1-containing complexes under salt conditions stabilized miRNA161 and miRNA173, immunoprecipitation (IP) of AGO1 complexes was performed, and both miRNAs were detected in the IP samples by northern hybridization. The results clearly indicated that miRNA161 and miRNA173 induced during salt stress were bound to AGO1 (Fig. 4B; Supplemental Fig. S9A). The role of AGO1 in the stabilization of selected miRNAs during salt stress was also demonstrated using a set of ago1 mutants (Kidner and Martienssen, 2005; Baumberger and Baulcombe, 2005). The results revealed that the stabilization effect of salt stress on miRNA161 and miRNA173 was abolished in the ago1-11 mutant, which contains a moderate AGO1 allele (Fig. 4C; Supplemental Fig. S9B). When null ago1 mutants were used, the levels of miRNA161 and miRNA173 markedly decreased under both stress and control conditions. However, we did not observe any overaccumulation of miRNA161 and miRNA173 that would be induced by stress (Supplemental Fig. S9C). These observations confirm the crucial role of AGO1 in the stabilization of miRNA161 and miRNA173.

The Expression Levels of MIR161 and MIR173 Are Not Regulated at the Level of the Initiation of Transcription during Salt Stress

To identify the MIRs that are regulated at the transcriptional level during salt stress, we constructed several Arabidopsis lines carrying the β-glucuronidase (GUS) coding sequence under the control of selected MIR gene promoters. As expected, we did not observe any changes in GUS activity when GUS expression was driven by the MIR161 and MIR173 promoters (Fig. 5A). In these two cases, we observed a negative correlation between the pri-miRNA and miRNA levels during salt stress (Fig. 2; Supplemental Fig. S7B). To ensure that the obtained results were not an effect of the high level of GUS enzyme stability, we also analyzed the level of GUS mRNA. The results did not reveal any changes between control and stressed plants (Fig. 5B). In parallel, we constructed and examined transgenic plants carrying the GUS coding sequence under the MIR163 and MIR829 promoters. The primary transcripts levels in both these MIR genes were higher in response to salt stress in our mirEX experiments. In contrast to the MIR161 and MIR173 promoters, in these two cases, we observed a significant increase in GUS activity in response to salt stress as well as elevated GUS mRNA levels, which were monitored by RT-qPCR (Supplemental Fig. S10). Thus, the results of the MIR promoter analyses indicated that MIR161 and MIR173 were not regulated in response to salt stress at the level of transcription initiation.

Figure 5.

Figure 5.

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Activity of MIR161 and MIR173 promoters in normal and salt stress conditions. A, Staining of Arabidopsis reporter lines expressing GUS under the control of the MIR161 and MIR173 promoters. B, GUS mRNA and pri-miRNA levels in Arabidopsis reporter lines expressing GUS under the control of MIR161 and MIR173 promoters in response to normal and salt stress conditions. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

MIR161 and MIR173 Are Regulated Cotranscriptionally

Another possible explanation for the underaccumulation of pri-miRNA161 and pri-miRNA173 after salt treatment could be an accelerated processing of these precursors into mature miRNAs. This process would result in decreased levels of miRNA precursor and increased amounts of mature molecules, as observed for miRNA161 and miRNA173. To test this hypothesis, we blocked the transcription with cordycepin and analyzed the levels of pri-miRNA161 and pri-miRNA173 under normal and stress conditions. The results obtained for full-length transcripts with a poly(A) tail indicated that there were no differences between the processing ratio of pri-miRNA161 and pri-miRNA173 in response to salt stress compared with control untreated plants (Fig. 6A). Equivalent results were obtained for pri-miRNA403, which was not affected during salt stress and served as a control in this experiment (Supplemental Figs. S4D and S7C). Surprisingly, different results were obtained when we analyzed the entire pool of transcripts from the MIR161 and MIR173 genes, and not only the polyadenylated ones (the cDNA samples used in this experiment were prepared with random primers): the half-life of the MIR161 and MIR173 transcripts was significantly shorter during salinity stress. In contrast, there were no changes in the stability of the control pri-miRNA403 (Fig. 6B). These results indicated that salt stress led to an increased amount of nonpolyadenylated MIR161 and MIR173 transcripts that displayed reduced stability. Thus, during salt stress, we observed an overaccumulation of nonpolyadenylated MIR161 and MIR173 transcripts. This result suggested that MIR161 and MIR173 could be regulated cotranscriptionally.

Figure 6.

Figure 6.

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Half-life of pri-miRNAs under normal and salt stress conditions. A, The half-life of full-length, poly(A)-tailed miRNA precursors. B, The half-life of the entire pool of MIR transcripts. RT-qPCR analyses of MIR161, MIR173, and MIR403 (used herein as a control) gene transcripts were conducted after blocking transcription with cordycepin. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

To confirm this hypothesis, we performed chromatin immunoprecipitation (ChIP) assays to examine the distribution of RNAPII on MIR161 and MIR173. In both cases, we observed that, under stress conditions, the level of total RNAPII decreased, but only in a gene body region and not within the MIR promoter sequences (Fig. 7). No significant changes were observed for the control MIR403 gene (Fig. 7). In parallel, we analyzed a set of transcriptionally regulated MIR genes (up-regulated MIR163, MIR168a, and MIR829). For MIR163, MIR168a, and MIR829, changes in RNAPII occupancy in the promoter region were detected as well as increased levels of total polymerase within the genes, indicating intense transcription of MIR163, MIR168a, and MIR829 under stress conditions (Supplemental Fig. S11). These observations are consistent with our original suggestion based on the results of the mirEX platform experiments, which showed that the expression of MIR163, MIR168a, and MIR829 is regulated at the level of transcription (Fig. 2A; Supplemental Figs. S4A and S7A).

Figure 7.

Figure 7.

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Distribution of RNA polymerase II across the MIR161, MIR173, and MIR403 genes under normal and salt stress conditions. ChIP was performed using antibodies against total RNAPII. Above each chart, gene structure is shown with black boxes representing pri-miRNA coding sequence; gray boxes, pre-miRNA coding sequence; white boxes, promoter regions; black lines, introns and regions downstream of MIR genes. Red lines show amplified regions. Above each gene structure, 0.5 kb scale is shown. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

AGO1 Cotranscriptionally Influences pri-miRNA Levels in Response to Salt Stress

In animal cells, AGO1 can regulate the expression of transcribed genes (Huang et al., 2013). This interaction may also affect RNAPII processivity and abolish splicing of selected alternative exons (Ameyar-Zazoua et al., 2012; Li et al., 2012b; Alló et al., 2014). There have also been some suggestions that, in Arabidopsis, AGO1 localization is not limited to the cytoplasm (Ye et al., 2012; Duan et al., 2012). Using immunolocalization and western-blot analysis, we showed that AGO1 is indeed localized in the nucleus of Arabidopsis cells (Fig. 8). To test our hypothesis regarding the potential involvement of AGO1 in the regulation of MIR161 and MIR173 expression, we performed stress experiments using the ago1-11 mutant. The results showed that salt stress still diminished the levels of pri-miRNA161 and pri-miRNA173 in the ago1-11 mutant (Fig. 9A), but, in comparison to wild-type plants, only a partial reduction in transcript levels was observed (Supplemental Fig. S12).

Figure 8.

Figure 8.

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Subcellular localization of AGO1. Immunolocalization of AGO1 in Arabidopsis mesophyll cells (A) and in isolated mesophyll cell nuclei (B). Scale bars = 5 μm. C, Detection of AGO1 (western) in total, cytoplasmic and nuclear fractions using both anti-AGO1 and anti-FLAG antibodies. For the fractionation of cell compartments, the AGO1:FLAG-expressing line (+) and wild-type plants (−) were used. Cy, Cytoplasm; N, nucleus; Nu, nucleolus.

Figure 9.

Figure 9.

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AGO1 is important for MIR161 and MIR173 gene expression in salt stress. A, The relative expression levels of pri-miRNA161, pri-miRNA173, and pri-miRNA403 under salt stress conditions in wild-type plants and the ago1-11 mutant. B, ChIP in the ago1-11 mutant using total RNAPII antibodies. C, Distribution of AGO1 on MIR161, MIR173, and MIR403 genes under standard and salt stress conditions. ChIP in the Arabidopsis AGO1:FLAG line using anti-FLAG antibodies. Data normalized to a no-FLAG control. Above each chart, gene structure is shown with black boxes representing pri-miRNA coding sequence; gray boxes, pre-miRNA coding sequence; white boxes, promoter regions; black lines, introns and regions downstream of MIR genes. Red lines show amplified regions. Above each gene structure, 0.5 kb scale is shown. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

We examined also the RNAPII distribution across MIR161 and MIR173 genes in the ago1-11 mutant under control and salt stress conditions. Similar to the results of the experiment performed on wild-type plants, after salt treatment of ago1-11 mutants the decreased level of RNAPII was detected (Fig. 9B) but the differences between stress and control conditions were significantly smaller than those observed previously in wild-type plants (Supplemental Fig. S13). In contrast, no change in the RNAPII distribution was observed on the MIR403 gene, which served as a salt nonresponsive gene control. (Supplemental Fig. S13). Surprisingly, we observed in ago1-11 the decrease of RNAPII level upstream of the transcription start sites of MIR161 and MIR173 genes (Supplemental Fig. S13A). This change of the RNAPII level at the promoter regions of MIR161 and MIR173 was not observed in the case of the control MIR403 gene.

AGO1 Accumulates on MIR161 and MIR173 Loci in Response to Salt Stress

The results showing that AGO1 could be involved in the regulation of MIR expression in salt stress encouraged us to examine the distribution of AGO1 across MIR161 and MIR173 as well as across the control MIR403 gene. The ChIP results revealed that the AGO1 protein localized on the MIR161 and MIR173 genes, and the level of chromatin-associated AGO1 increased in response to salt treatment, mainly in the stem-loop miRNA coding region (Fig. 9C). This phenomenon was not detected in MIR403 that demonstrated no changes in expression during salinity stress (Fig. 9C). Additionally, the AGO1 ChIP experiment performed in the presence of RNaseA clearly indicated that the interaction between AGO1 and chromatin was mediated by RNA (Fig. 10). To answer the question if AGO1 association with chromatin is limited only to MIR161 and MIR173 genes, we tested whether the AGO1 protein can be localized on other than MIR161 and MIR173 miRNA genes, as well as on the protein gene encoded ACTIN II. The results obtained indicated that under salt stress AGO1 appears also in close proximity to MIR157c and MIR827 (Supplemental Fig. S14A). For both these MIRs, similar to MIR161 and MIR173, a negative correlation in the expression levels of pri-miRNA and mature miRNA has been found under stress conditions (Supplemental Fig. S4, B and C). For MIRs that show positive correlations in the expression levels of pri-miRNAs and mature miRNAs under stress conditions (MIR163, MIR165a, and MIR829), AGO1 was not detected in this ChIP experiment (Supplemental Fig. S14B). However, MIR168A, which belongs to the same group of miRNA genes, shows enrichment of AGO1 at this locus but in the stress-independent manner (Supplemental Fig. S14A). No AGO1 association was detected in the case of ACTIN II.

Figure 10.

Figure 10.

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Association of AGO1 with chromatin in wild-type, hyl1-2, and cpl1-7 mutant plants monitored under standard and salt stress conditions. A, Association of AGO1 with the MIIR161 gene. B, Association of AGO1 with the MIR173 gene. ChIP was performed using antibodies against AGO1. For the experiment, primers amplifying pre-miRNA coding region were used. For wild-type plants, ChIP with and without RNase A was conducted. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

Because miRNA173 is involved in biogenesis of ta-siRNAs that derived from the transcripts of TAS1 and TAS2 genes, we tested also the localization of AGO1 on the TAS loci. In these ChIP experiments the AGO1 protein was detected on TAS1a/b/c and TAS2 but not on TAS3 transcripts. It is known that TAS3 transcripts are not cleaved by the AGO1/miRNA173 complex, but by the AGO7-containing complex loaded with miRNA390 (Montgomery et al., 2008).

HYL1 Plays a Special Role in the Regulation of MIR161 and MIR173 Transcription in Response to Stress

We were also interested in determining whether AGO1 requires loading with miRNAs to interact with chromatin. Therefore, we evaluated the salt stress response in Arabidopsis mutants with unpaired miRNA biogenesis, and the levels of mature miRNAs decreased significantly. First, we used the hyl1-2 mutant, which completely lacks the HYL1 protein, a dsRNA binding protein involved in miRNA biogenesis (Vazquez et al., 2004; Park et al., 2002; Han et al., 2004). HYL1 is also known to interact with AGO1 (Fang and Spector, 2007) and is involved not only in pri-miRNA processing but also in the loading of AGO1 with miRNAs (Eamens et al., 2009). During salt stress, we did not observe any changes in miRNA161 and miRNA173 levels in the hyl1-2 mutant (Supplemental Fig. S15). It is worth noting that the level of AGO1 mRNA in the hyl1 mutant increased because HYL1 is needed for the proper accumulation of miRNA168, which targets AGO1 mRNA (Vaucheret et al., 2004). Interestingly, the level of AGO1 protein in the hyl1-2 mutant was lower than that in wild-type plants and remained unchanged under salinity conditions, in contrast to wild type in which the level of AGO1 increased after NaCl treatment (Supplemental Fig. S16). In the hyl1-2 mutant, we did not observe any reduction of the pri-miRNA161 and pri-miRNA173 in response to salt treatment (Fig. 11A; Supplemental Fig. S17). Moreover, in contrast to wild type (Fig. 7), no differences in RNAPII occupancy of MIR161 and MIR173 were detected in hyl1-2 mutant plants during salt stress (Fig. 11B). Finally, ChIP assays in the hyl1-2 background utilizing antibodies against AGO1 revealed that the decreased miRNAs levels observed in hyl1-2 disrupted the interaction of AGO1 with chromatin in response to salt stress (Fig. 10). We also tested two other Arabidopsis miRNA biogenesis mutants: the double mutant cbp20cbp80 (cbc), which lacks both subunits of the CBC; and se-1, which contains a truncated and not fully active version of SE, a key factor in miRNA biogenesis (Dong et al., 2008; Laubinger et al., 2008). In both mutants, similar to hyl1-2, an underaccumulation of miRNAs was observed, while their precursors displayed an overaccumulation. In contrast to the hyl1-2 mutant in which the levels of miRNA161 and miRNA173 as well as pri-miRNA161 and pri-miRNA173 remained unchanged in response to salt treatment, only partial inhibition of the salt stress response was observed in cbc and se-1 mutants (Supplemental Fig. S18). These findings indicate that HYL1 plays an important role in the regulation of miRNA expression in salinity, and this role can be associated not only with miRNA biogenesis but also with its activity in the loading of AGO1 with miRNAs.

Figure 11.

Figure 11.

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Hyl1 plays a role in the regulation of MIR161 and MIR173 transcription in salt stress. A, Relative expression levels of pri-miRNA161, pri-miRNA173, and pri-mRNA403 under salt stress conditions in the hyl1-2 mutant. B, ChIP in the hyl1-2 mutant using total RNAPII antibodies. Above each chart, gene structure is shown with black boxes representing pri-miRNA coding sequence; gray boxes, pre-miRNA coding sequence; white boxes, promoter regions; and black lines, introns and regions downstream of MIR genes. Red lines show amplified regions. Above each gene structure, 0.5 kb scale is shown. The values presented in the charts are the mean ± sd (n = 3). * t test P value ≤ 0.05; ** t test P value ≤ 0.01.

CPL1 Influences the MIR161 and MIR173 Expression in Response to Salinity

It is known that during miRNA biogenesis, HYL1 is dephosphorylated by CPL1 phosphatase, and that this modification of HYL1 protein stimulates its activity (Manavella et al., 2012). Interestingly, CPL1 was originally described as a phosphatase that is important for dephosphorylation of the RNAPII C-terminal domain (CTD) at Ser-5 (Koiwa et al., 2004; Jiang et al., 2013). This phosphorylation of CTD is required for cap formation, the transition from the initiation to the elongation phase and proper poly(A) site selection (Jiang et al., 2013). It has been suggested that the phosphorylation of CTD Ser-5 is an indication of enzyme pausing during transcription (Alexander et al., 2010; Dolata et al., 2015). Therefore, we performed the stress experiments in the cpl1-7 mutant. The results demonstrated that the levels of pri-miRNA161 and pri-miRNA173 were down-regulated in cpl1-7 (Supplemental Fig. S19A), as previously described for other miRNA precursors (Manavella et al., 2012). However, compared with wild-type plants, we observed only a partial reduction of the pri-miRNA161 and pri-miRNA173 levels in response to salt stress (Supplemental Fig. S19B). The ChIP experiments showed that RNAPII accumulates on the MIR genes that were tested in the cpl1-7 mutant (Supplemental Fig. S19C), suggesting reduced processivity and/or pausing of polymerase in cpl1-7. However, in the cpl1-7 genetic background, the lower accumulation of RNAPII on MIR161 and MIR173 after salt treatment was still observed, suggesting that, in the absence of CPL1, the AGO1 protein is still capable of impacting MIR161 and MIR173 during stress conditions, and this interaction leads to a reduction of pri-miRNA161 and pri-mRNA173 levels. Similarly, compared with wild-type plants, the AGO1 ChIP analysis in the cpl1-7 background showed decreased but still significant AGO1 binding to the MIR161 and MIR173 genes during salt stress (Fig. 10).

DISCUSSION

AGO1 is described as a key component of the plant RISC complex, acting in the cytoplasm by cutting mRNA targets or inhibiting their translation (Llave et al., 2002; Reinhart and Bartel, 2002; Palatnik et al., 2003; Li et al., 2013). This well-known function of AGO1 is crucial for the final effect of miRNAs as negative regulators of gene expression. In this study, we showed that Arabidopsis AGO1 plays an important role in response to environmental conditions not only by participating in targeting mRNA cleavage but also in the stabilization of salinity stress-induced miRNA molecules. Our results revealed that the AGO1 protein also functions in the nucleus of plant cells, where it affects the transcription of certain MIR genes. Thus, through the study of salinity stress, we demonstrated that AGO1 in Arabidopsis is involved in the posttranscriptional regulation of stress-induced miRNAs via their stabilization in the cytoplasm and cotranscriptionally regulates the expression of selected MIRs in the nucleus.

AGO1 Stabilizes Selected miRNA Molecules in Response to Salt Stress

The levels of 40% of all Arabidopsis pri-miRNAs were changed during salt stress. In some cases, the increased levels of MIR gene transcription resulted in higher miRNA production, thus leading to more efficient cleavage or translation inhibition of the target mRNAs. During salt stress, we observed this phenomenon as most likely due to the transcriptional regulation of miRNA expression in many of the salt-responsive miRNAs. However, in some cases, e.g. miRNA161 and miRNA173, the decreased level of primary precursors correlated with a higher accumulation of mature miRNA molecules. It has already been suggested that these negative correlations between the levels of pri-miRNAs and their cognate mature miRNAs result from many different environmental stresses, including salinity (Barciszewska-Pacak et al., 2015); however, the molecular mechanism underlying such nontranscriptional regulation of miRNA expression remains unknown. Interestingly, after a 24-h-long treatment of Arabidopsis seedlings with 250 mm NaCl, the levels of miRNA161 and miR173 did not increase, indicating that the salinity-induced accumulation of miRNA161 and miRNA173 observed is part of the rapid response of plants to this environmental stimulus (Barciszewska-Pacak et al., 2015).

The accumulation of selected miRNAs under salt stress was limited only to functional miRNA molecules and not their complementary miRNAs*, indicating that there is a specific mechanism involved in such salt-induced accumulation of selected miRNAs. Our data showed that a lack of AGO1 led to a dramatic decrease in miRNA levels, supporting the importance of AGO1 in general miRNA stabilization. The results obtained for the mutated version of AGO1 (the stress experiments performed in the ago1-11 mutant and in other ago1 mutants) support its important role in the stabilization of miRNA161 and miRNA173 in response to salt treatment. Our data showed that during salinity stress, AGO1 bound to and stabilized both of these miRNAs, leading to effective cleavage of their target sequences. A similar stabilization effect of AGO1 binding has been observed for miR168, which targets AGO1 mRNA (Vaucheret et al., 2006; Vaucheret, 2009).

Interestingly, the ago1-11 mutant shows a delayed germination phenotype under salt stress conditions (Supplemental Fig. S20). These results support the idea of the involvement of AGO1 in the plant physiological response to salinity. A similar stress-related phenotype has already been described for another ago1 mutant, ago1-27 (Li et al., 2012b). However, no difference was observed between wild-type and ago1-11 plants when the expression of three salt-stress-related genes, ABF3, LEA4-5, and TSPO, was determined before and after NaCl treatment: in both wild-type and ago1-11 plants, overexpression of the stress marker genes was observed (Supplemental Fig. S21). Thus, the results obtained indicate that AGO1 is important for the proper response of plants to salinity but it is involved in a specific pathway(s), and does not participate in all salt stress-induced molecular responses.

AGO1 Acts Cotranscriptionally on MIR161 and MIR173 Genes

Our data demonstrate that AGO1, in addition to its function in the cytoplasm, is also present in the plant cell nucleus. We have shown that under salinity stress, nuclear AGO1 is associated with chromatin within the regions that encode pre-miRNA161 and pre-miRNA173. This stress-induced accumulation of AGO1 at MIR161 and MIR173 loci correlates with the decreased levels of RNAPII in the bodies of both MIRs, but not in their promoter regions. This finding indicates that many RNAPII molecules are likely to pause under stress conditions during the synthesis of pri-miRNA161 and pri-miRNA173. The AGO1 located at MIR161 and MIR173 loci may lead to polymerase pausing and induce its detachment. These premature terminations of transcription can explain the presence of nonpolyadenylated, incomplete MIR161 and MIR173 transcripts in plants growing under stress conditions, as detected in our studies. It is possible that the AGO1/miRNA complex interacts with a newly transcribed pri-miRNA via the miRNA complementary miRNA* sequence and cleaves it cotranscriptionally. Previously published data, obtained from global identification of miRNA targets, showed some MIR transcripts cleaved in this way (German et al., 2008). However, in this study we were unable to detect any specifically cleaved products of the MIR161 and MIR173 primary transcripts by rapid amplification of cDNA ends. Our ChIP analyses revealed that the accumulation of AGO1 within the MIR161 and MIR173 genes was clearly RNase-sensitive, suggesting that the analyzed interactions were mediated by RNA. Thus, although RNA is important for the interaction of AGO1 with chromatin on the MIR161 and MIR173 genes, the cotranscriptional regulation of their expression by this binding does not appear to be achieved by miRNA-mediated cleavage of nascent transcripts. There is also the possibility that the level of miRNA-induced cleaved fragments was too low to be detected using the applied method. The salt stress-induced association of AGO1 with other MIR loci (MIR157c and MIR827) was also found in this study. Moreover, under stress conditions AGO1 accumulates on the TAS loci, the transcripts of which are the targets of salt-induced miRNA173. These observations, however, require further molecular analyses to uncover the functional meaning of the AGO1 accumulation on the TAS1 and TAS2 loci.

The nuclear functions of animal AGO1/2 proteins have been previously described. Two genome-wide surveys in Drosophila melanogaster have shown that AGO2 is involved in nuclear processes that regulate alternative splicing as well as the transcription of target genes (Cernilogar et al., 2011; Taliaferro et al., 2013). In addition, a study in human cancer cells suggested that AGO1 interacts with RNAPII and binds to transcriptionally active promoters (Huang et al., 2013). Moreover, a more recent genome-wide analysis revealed that human AGO1 binds preferentially to active transcriptional enhancers and that this association is mediated by the RNAs that are transcribed from these enhancers, the eRNAs. Interestingly, the interaction of AGO1 with enhancers does not appear to regulate the transcription of neighboring genes but of alternative and constitutive splicing (Li et al., 2012b). Thus, in animal cells, the nuclear role of AGO1/2 proteins in the regulation of transcription and splicing has been well documented. To this list of nuclear functions of AGOs, our results provide new information regarding the cotranscriptional regulation of the biogenesis of miRNA in plants.

A Special Role of HYL1 in the Regulation of MIR161 and MIR173 Genes in Response to Salt Treatment

Our results suggest that the AGO1 protein, which is involved in the cotranscriptional regulation of the expression of selected MIR genes, must be loaded with miRNAs. In cbc, se-1, and cpl1-7 Arabidopsis mutants, only a partial reduction of the levels of pri-miR161 and pri-miRNA173 was observed. In all of these Arabidopsis mutants, the level of miRNAs was significantly reduced, suggesting a correlation between the accumulation of miRNA and the involvement of AGO1 in the regulation of MIR161 and MIR173 gene expression. However, this novel nuclear role of AGO1 was completely abolished in another miRNA biogenesis mutant, hyl1-2. This result indicated that HYL1 is a crucial element in the regulation of MIR161 and MIR173 expression in response to salinity. It has been shown that the HYL1 protein is involved not only in the stimulation of cleavage catalyzed by DCL1 during miRNA biogenesis but also in the loading of AGO1 with miRNAs (Eamens et al., 2009). Therefore, the effect of the absence of HYL1 in the hyl1-2 mutant is substantially stronger than that in mutants of other proteins that participate in pri-miRNA processing. Taken together, our results suggest that AGO1 performs its functions in the nucleus via the cotranscriptional regulation of salt-responsive MIRs, and that AGO1 involved in this regulation is most likely loaded with miRNAs.

A Model of the Cotranscriptional Regulation of MIR Gene Expression by AGO1

Based on our results, we propose a model of the cotranscriptional regulation of MIR161 and MIR173 expression (Fig. 12). During salinity stress, the levels of miRNA161 and miRNA173 increased mostly via the stabilization of these two miRNAs by AGO1. In the cytoplasm, miRNA161 and miRNA173, after incorporation into the RISC complex, perform their functions by altering the levels of the TAS transcript targets. A portion of AGO1, which is most likely loaded with miRNA161 and miRNA173, functions in the nucleus to control the expression of MIR161 and MIR173 cotranscriptionally. In our model, the AGO1/miRNA complex interacts with a newly synthesized pri-mRNA and causes the premature disassembly of RNAPII from the DNA template. The molecular mechanism underlying the communication between AGO1 and RNAPII, as well as AGO1 specificity for particular MIR genes, is not clear and requires further investigation.

Figure 12.

Figure 12.

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A model of the cotranscriptional regulation of MIR gene expression by AGO1.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) T87 cells were cultivated in Gamborg B5 medium (Sigma-Aldrich) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma-Aldrich) to a final concentration of 0.1 μg/L. The cells were grown in a shaker (Innova44; New Brunswick Scientific) at 22°C with constant light (120–200 μM m2/s) and rotation (120 rpm). Wild-type Col-0 and Ler plants, ago1-11, ago1-36, ago1-41, ago1-42, ago1-101, hyl1-2, se-1, cpl1-7, and cbp20cbp80 mutants (Kidner and Martienssen et al., 2005; Baumberger and Baulcombe, 2005; Song et al., 2007; Manavella et al., 2012) and the FLAG:AGO1 line (Baumberger and Baulcombe, 2005) were used in these studies. Fourteen-d-old Arabidopsis seedlings were cultivated on half-strength Murashige and Skoog medium (1/2 MS; Duchefa Biochemie) solidified with 1% agar (Sigma-Aldrich). The seeds were sterilized with chlorine fumes and sown on plates under sterile conditions. The plates were covered with parafilm and kept in the dark for 48 h at 4°C. The samples were subsequently transferred into a plant growth chamber MLR 350 (Sanyo). A 16-h/8-h photoperiod with a light intensity 120–200 μM m2/s was applied at 22°C. Salt stress was induced by the addition of NaCl to a final concentration of 250 mm to both T87 cell cultures or to flasks with liquid 1/2 MS medium containing Arabidopsis seedlings that had been transferred earlier from plates to the flasks. Salt stress was conducted for 60 min (T87) or 120 min (seedlings).

Generation of Transgenic Lines

The promoters of MIR161 (2462 bp), MIR163 (2004 bp), MIR173 (2268 bp), and MIR829 (2536 bp) were amplified by PCR (oligonucleotides are listed in Supplemental Table S2) and cloned into pENTR/D-TOPO (Thermo Fisher Scientific) using NotI and AscI restriction sites. All constructs used were verified by sequencing. For expression in plants, pMDC163 Gateway binary vectors were used (Curtis and Grossniklaus, 2003). Transgenes were introduced into Arabidopsis Col-0 plants via Agrobacterium-mediated floral dip transformation (Clough and Bent, 1998).

RNA Isolation and Analysis

Total RNA from T87 cells or Arabidopsis seedlings was isolated using TRIzol reagent (Thermo Fisher Scientific) or the Direct-zol RNA MiniPrep kit (Zymo Research). RNA was treated with Turbo DNase (Thermo Fisher Scientific) before reverse transcription with oligo dT(18) (Thermo Fisher Scientific) or random primers (Thermo Fisher Scientific), and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Small RNAs were analyzed as previously described in Szarzynska et al. (2009).

RT-qPCR Analyses

RT-qPCR was performed as previously described in Sierocka et al. (2011). The data were normalized to the GAPDH (At1g13440) transcript level and are presented as the fold change or relative expression level (log10)+10 to demonstrate the expression profile within a positive data range.

RNA Half-Life Measurements

Half-lives were determined as previously described in Seeley et al. (1992) with the following modifications. Arabidopsis seedlings were transferred into incubation buffer (Seeley et al., 1992), and 3′-deoxyadenosine (cordycepin; Sigma-Aldrich) was added to a final concentration of 0.6 mm (time 0). Tissue samples were harvested at regular intervals. Total RNA was isolated, treated with Turbo DNase (Thermo Fisher Scientific), and reverse-transcribed (with oligo dT(18) or random primers) for qPCR.

ChIP

ChIP was performed as previously described in Dolata et al. (2015) using 2 μg of each antibody per IP: anti-RNAPII (AS11 1804; Agrisera), anti-AGO1 (AS09 527; Agrisera), and 30 μL of ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich). The RNase+ nuclear extract sample was treated with 2.5 µg of RNase A (Thermo Fisher Scientific) during IP. In the experiments in which anti-FLAG antibody was used, wild-type (Col-0) plants served as a background control. In the experiments, with native antibodies no-antibody samples were used as a background control.

RNA IP

The nuclear fraction used for RNA IP was obtained as described for the ChIP experiments. RNA-specific steps were performed as described by Rowley et al. (2013). AGO1 complexes were immunoprecipitated using ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich). RNA was used directly in the northern hybridization. The supernatant from the first step of the nuclear isolation was used as a cytoplasmic fraction. In the experiments in which anti-FLAG antibody was used, wild-type (Col-0) plants served as a background control.

Immunolocalization

The leaves used for the immunofluorescence analysis were fixed with 4% formaldehyde (Polyscience) for 1 h and placed in a vacuum for 1 h to remove air from the intracellular spaces. The plant material was washed five times in PBS (pH 7.2) for 3 min according to the protocol described by Delenko et al. (2015). The leaves were then gently homogenized in a Potter homogenizer to obtain single cells. Triton X-100 (Sigma-Aldrich) in PBS, pH 7.2 (1:1000) was used to permeabilize the cell membrane. After this treatment, the material was washed three times for 5 min each in PBS, pH 7.2. The samples were centrifuged for 3.5 min at 0.1g. After blocking with 1% BSA (Sigma-Aldrich) in PBS, pH 7.2 for 30 min, the cell suspensions were incubated with rabbit anti-AGO1 (AS09 527; Agrisera) primary antibody in 1% BSA in PBS, pH 7.2 (1:200) overnight at 4°C. After three washes in PBS, pH 7.2, the samples were incubated with goat anti-rabbit secondary antibody (Thermo Fisher Scientific) labeled with Alexa Fluor 488 (1:500). This incubation was performed in 1% BSA in PBS, pH 7.2 for 1 h at 37°C. In the control experiment, all of the steps were performed with omission of the primary antibodies. After three washes in PBS, pH 7.2, the DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) at 2 μg/mL. The samples were analyzed using a PCM 2000-Eclipse TE 300 confocal microscope (Nikon).

Western Blot

Protein extracts were separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (PVDF; Millipore), and analyzed by western blot using antibodies at the indicated dilutions: anti-H3 (1791; Abcam) at 1:1000; anti-Actin II (0869100; MP Biomedicals) at 1:5000; AGO1 (AS09 527; Agrisera) at 1:10,000; and anti-FLAG at 1:2000 (Sigma-Aldrich).

Statistics

Statistical tests were performed using MS-Excel 2007 (Microsoft). Student’s t test was applied.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. R. Martienssen for providing ago1 mutants seeds and Dr. D. Baulcombe for providing the FLAG:AGO1 line.

Footnotes

[OPEN]

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References

  1. Alexander RD, Innocente SA, Barrass JD, Beggs JD (2010) Splicing-dependent RNA polymerase pausing in yeast. Mol Cell 40: 582–593 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alló M, Agirre E, Bessonov S, Bertucci P, Gómez Acuña L, Buggiano V, Bellora N, Singh B, Petrillo E, Blaustein M, Miñana B, Dujardin G, et al. (2014) Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells. Proc Natl Acad Sci USA 111: 15622–15629 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau JC, Mathieu J, Hamiche A, et al. (2012) Argonaute proteins couple chromatin silencing to alternative splicing. Nat Struct Mol Biol 19: 998–1004 [DOI] [PubMed] [Google Scholar]
  4. Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P, Pacak AM, Vazquez F, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2015) Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses. Front Plant Sci 6: 410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102: 11928–11933 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bielewicz D, Dolata J, Zielezinski A, Alaba S, Szarzynska B, Szczesniak MW, Jarmolowski A, Szweykowska-Kulinska Z, Karlowski WM (2012) mirEX: a platform for comparative exploration of plant pri-miRNA expression data. Nucleic Acids Res 40: D191–D197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bielewicz D, Kalak M, Kalyna M, Windels D, Barta A, Vazquez F, Szweykowska-Kulinska Z, Jarmolowski A (2013) Introns of plant pri-miRNAs enhance miRNA biogenesis. EMBO Rep 14: 622–628 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cernilogar FM, Onorati MC, Kothe GO, Burroughs AM, Parsi KM, Breiling A, Lo Sardo F, Saxena A, Miyoshi K, Siomi H, Siomi MC, Carninci P, et al. (2011) Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila. Nature 480: 391–395 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chatterjee S, Grosshans H (2009) Active turnover modulates mature microRNA activity in Caenorhabditis elegans. Nature 461: 546–549 [DOI] [PubMed] [Google Scholar]
  10. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 [DOI] [PubMed] [Google Scholar]
  11. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133: 462–469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Daszkowska-Golec A, Wojnar W, Rosikiewicz M, Szarejko I, Maluszynski M, Szweykowska-Kulinska Z, Jarmolowski A (2013) Arabidopsis suppressor mutant of abh1 shows a new face of the already known players: ABH1 (CBP80) and ABI4-in response to ABA and abiotic stresses during seed germination. Plant Mol Biol 81: 189–209 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Delenko K, Niedojadlo J, Labedzka A, Wisniewska E, Bednarska-Kozakiewicz E (2015) Dedifferentiation of Arabidopsis thaliana cells is accompanied by a strong decrease in RNA polymerase II transcription activity and poly(A+) RNA and 25S rRNA eradication from the cytoplasm. Protoplasma 252: 537–546 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dolata J, Guo Y, Kołowerzo A, Smoliński D, Brzyżek G, Jarmołowski A, Świeżewski S (2015) NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis. EMBO J 34: 544–558 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dong Z, Han MH, Fedoroff N (2008) The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci USA 105: 9970–9975 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duan CG, Fang YY, Zhou BJ, Zhao JH, Hou WN, Zhu H, Ding SW, Guo HS (2012) Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the Cucumber mosaic virus 2b protein. Plant Cell 24: 259–274 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eamens AL, Smith NA, Curtin SJ, Wang MB, Waterhouse PM (2009) The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15: 2219–2235 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fang Y, Spector DL (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 17: 818–823 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, et al. (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26: 941–946 [DOI] [PubMed] [Google Scholar]
  20. Han MH, Goud S, Song L, Fedoroff N (2004) The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci USA 101: 1093–1098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang V, Zheng J, Qi Z, Wang J, Place RF, Yu J, Li H, Li LC (2013) Ago1 Interacts with RNA polymerase II and binds to the promoters of actively transcribed genes in human cancer cells. PLoS Genet 9: e1003821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hugouvieux V, Kwak JM, Schroeder JI (2001) An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis. Cell 106: 477–487 [DOI] [PubMed] [Google Scholar]
  23. Jiang J, Wang B, Shen Y, Wang H, Feng Q, Shi H (2013) The Arabidopsis RNA binding protein with K homology motifs, SHINY1, interacts with the C-terminal domain phosphatase-like 1 (CPL1) to repress stress-inducible gene expression. PLoS Genet 9: e1003625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kai ZS, Pasquinelli AE (2010) MicroRNA assassins: factors that regulate the disappearance of miRNAs. Nat Struct Mol Biol 17: 5–10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kidner CA, Martienssen RA (2005) The role of ARGONAUTE1 (AGO1) in meristem formation and identity. Dev Biol 280: 504–517 [DOI] [PubMed] [Google Scholar]
  26. Kmieciak M, Simpson CG, Lewandowska D, Brown JW, Jarmolowski A (2002) Cloning and characterization of two subunits of Arabidopsis thaliana nuclear cap-binding complex. Gene 283: 171–183 [DOI] [PubMed] [Google Scholar]
  27. Koiwa H, Hausmann S, Bang WY, Ueda A, Kondo N, Hiraguri A, Fukuhara T, Bahk JD, Yun DJ, Bressan RA, Hasegawa PM, Shuman S (2004) Arabidopsis C-terminal domain phosphatase-like 1 and 2 are essential Ser-5-specific C-terminal domain phosphatases. Proc Natl Acad Sci USA 101: 14539–14544 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kreps JA, Wu Y, Chang HS, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130: 2129–2141 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kruszka K, Pacak A, Swida-Barteczka A, Stefaniak AK, Kaja E, Sierocka I, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2013) Developmentally regulated expression and complex processing of barley pri-microRNAs. BMC Genomics 14: 34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kruszka K, Pieczynski M, Windels D, Bielewicz D, Jarmolowski A, Szweykowska-Kulinska Z, Vazquez F (2012) Role of microRNAs and other sRNAs of plants in their changing environments. J Plant Physiol 169: 1664–1672 [DOI] [PubMed] [Google Scholar]
  31. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101: 12753–12758 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laubinger S, Sachsenberg T, Zeller G, Busch W, Lohmann JU, Rätsch G, Weigel D (2008) Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc Natl Acad Sci USA 105: 8795–8800 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Li B, Takahashi D, Kawamura Y, Uemura M (2012a) Comparison of plasma membrane proteomic changes of Arabidopsis suspension-cultured cells (T87 Line) after cold and ABA treatment in association with freezing tolerance development. Plant Cell Physiol 53: 543–554 [DOI] [PubMed] [Google Scholar]
  34. Li S, Liu L, Zhuang X, Yu Y, Liu X, Cui X, Ji L, Pan Z, Cao X, Mo B, Zhang F, Raikhel N, et al. (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153: 562–574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li W, Cui X, Meng Z, Huang X, Xie Q, Wu H, Jin H, Zhang D, Liang W (2012b) Transcriptional regulation of Arabidopsis MIR168a and argonaute1 homeostasis in abscisic acid and abiotic stress responses. Plant Physiol 158: 1279–1292 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ludwików A, Cieśla A, Kasprowicz-Maluśki A, Mituła F, Tajdel M, Gałgański Ł, Ziółkowski PA, Kubiak P, Małecka A, Piechalak A, Szabat M, Górska A, et al. (2014) Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis. Mol Plant 7: 960–976 [DOI] [PubMed] [Google Scholar]
  38. Manavella PA, Hagmann J, Ott F, Laubinger S, Franz M, Macek B, Weigel D (2012) Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151: 859–870 [DOI] [PubMed] [Google Scholar]
  39. Meng Y, Shao C, Wang H, Chen M (2011) The regulatory activities of plant microRNAs: a more dynamic perspective. Plant Physiol 157: 1583–1595 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133: 128–141 [DOI] [PubMed] [Google Scholar]
  41. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425: 257–263 [DOI] [PubMed] [Google Scholar]
  42. Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102: 3691–3696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12: 1484–1495 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Raczynska KD, Stepien A, Kierzkowski D, Kalak M, Bajczyk M, McNicol J, Simpson CG, Szweykowska-Kulinska Z, Brown JWS, Jarmolowski A (2014) The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res 42: 1224–1244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Reinhart BJ, Bartel DP (2002) Small RNAs correspond to centromere heterochromatic repeats. Science 297: 1831. [DOI] [PubMed] [Google Scholar]
  46. Rowley MJ, Böhmdorfer G, Wierzbicki AT (2013) Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana. Methods 63: 160–169 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sasaki Y, Takahashi K, Oono Y, Seki M, Yoshida R, Shinozaki K, Uemura M (2008) Characterization of growth-phase-specific responses to cold in Arabidopsis thaliana suspension-cultured cells. Plant Cell Environ 31: 354–365 [DOI] [PubMed] [Google Scholar]
  48. Schwab R, Speth C, Laubinger S, Voinnet O (2013) Enhanced microRNA accumulation through stemloop-adjacent introns. EMBO Rep 14: 615–621 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Seeley KA, Byrne DH, Colbert JT (1992) Red light-independent instability of oat phytochrome mRNA in vivo. Plant Cell 4: 29–38 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sierocka I, Rojek A, Bielewicz D, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2011) Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort Pellia endiviifolia. Gene 485: 53–62 [DOI] [PubMed] [Google Scholar]
  51. Sokol A, Kwiatkowska A, Jerzmanowski A, Prymakowska-Bosak M (2007) Up-regulation of stress-inducible genes in tobacco and Arabidopsis cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications. Planta 227: 245–254 [DOI] [PubMed] [Google Scholar]
  52. Song L, Han MH, Lesicka J, Fedoroff N (2007) Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 104: 5437–5442 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Szarzynska B, Sobkowiak L, Pant BD, Balazadeh S, Scheible WR, Mueller-Roeber B, Jarmolowski A, Szweykowska-Kulinska Z (2009) Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs. Nucleic Acids Res 37: 3083–3093 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Taliaferro JM, Aspden JL, Bradley T, Marwha D, Blanchette M, Rio DC (2013) Two new and distinct roles for Drosophila Argonaute-2 in the nucleus: alternative pre-mRNA splicing and transcriptional repression. Genes Dev 27: 378–389 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: 49–63 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Várallyay E, Válóczi A, Agyi A, Burgyán J, Havelda Z (2010) Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation. EMBO J 29: 3507–3519 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Vaucheret H. (2009) AGO1 homeostasis involves differential production of 21-nt and 22-nt miR168 species by MIR168a and MIR168b. PLoS One 4: e6442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vaucheret H, Mallory AC, Bartel DP (2006) AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1. Mol Cell 22: 129–136 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vaucheret H, Vazquez F, Crété P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18: 1187–1197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crété P (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16: 69–79 [DOI] [PubMed] [Google Scholar]
  61. Winter J, Diederichs S (2011) Argonaute proteins regulate microRNA stability: increased microRNA abundance by Argonaute proteins is due to microRNA stabilization. RNA Biol 8: 1149–1157 [DOI] [PubMed] [Google Scholar]
  62. Wu X, Shi Y, Li J, Xu L, Fang Y, Li X, Qi Y (2013) A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis. Cell Res 23: 645–657 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yamada H, Koizumi N, Nakamichi N, Kiba T, Yamashino T, Mizuno T (2004) Rapid response of Arabidopsis T87 cultured cells to cytokinin through His-to-Asp phosphorelay signal transduction. Biosci Biotechnol Biochem 68: 1966–1976 [DOI] [PubMed] [Google Scholar]
  64. Ye R, Wang W, Iki T, Liu C, Wu Y, Ishikawa M, Zhou X, Qi Y (2012) Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes. Mol Cell 46: 859–870 [DOI] [PubMed] [Google Scholar]
  65. Yu B, Bi L, Zheng B, Ji L, Chevalier D, Agarwal M, Ramachandran V, Li W, Lagrange T, Walker JC, Chen X (2008) The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci USA 105: 10073–10078 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zielezinski A, Dolata J, Alaba S, Kruszka K, Pacak A, Swida-Barteczka A, Knop K, Stepien A, Bielewicz D, Pietrykowska H, Sierocka I, Sobkowiak L, et al. (2015) mirEX 2.0—an integrated environment for expression profiling of plant microRNAs. BMC Plant Biol 15: 144. [DOI] [PMC free article] [PubMed] [Google Scholar]

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