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- © 2017 American Society of Plant Biologists. All rights reserved.
Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3′ ends that map to 5′-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3′ ends within protein-coding regions and introns were less stable than mRNAs that end at 3′-UTR poly(A) sites. Additionally, these RNA isoforms were underrepresented in polysomes isolated from control and hypoxic plants. By contrast, mRNA isoforms with 3′ ends that lie within annotated 5′-UTRs were overrepresented in polysomes and were as stable as canonical mRNA isoforms. These results indicate that the generation of noncanonical mRNA isoforms is an important feature of the abiotic stress response. The finding that several noncanonical mRNA isoforms are relatively unstable suggests that the production of non-stop and intronic mRNA isoforms may represent a form of negative regulation in plants, providing a conceptual link with mechanisms that generate these isoforms (such as alternative polyadenylation) and RNA surveillance.
A major challenge of modern agriculture is to maintain crop production under adverse environmental conditions. Understanding the different mechanisms by which plants adapt and respond to environmental stresses may lead to novel strategies for crop improvement. Flooding, a compound abiotic stress that often leads to cellular hypoxia and energy depletion, is responsible for significant crop damage and yield losses in most crops (Bailey-Serres and Voesenek, 2008; Bailey-Serres et al., 2012b; Bailey-Serres and Colmer, 2014; Voesenek et al., 2016). The response of plants to hypoxic stress is highly complex and involves changes in mRNA and protein levels and subsequent biochemical and physiological adaptations.
Posttranscriptional regulation plays an important role in the control of gene expression. One mode of control that is accomplished through RNA processing is alternative polyadenylation (APA) (Di Giammartino et al., 2011; Lutz and Moreira, 2011; Shi, 2012; Akman and Erson-Bensan, 2014; de Klerk and 't Hoen, 2015; Mayr, 2016). Genome-wide studies in several eukaryotes have found that 10 to 50% of genes encode transcripts with multiple poly(A) sites (Tian et al., 2005; Nagalakshmi et al., 2008; Wu et al., 2011; Sherstnev et al., 2012). Poly(A) sites can occur at any position during the transcription of a gene and effectively determine protein length or influence mRNA stability, translation, and/or transport (Lutz and Moreira, 2011; Wu et al., 2011). APA may occur through differential selection of polyadenylation signals in the 3′-untranslated region (UTR) of pre-mRNAs. This results in the production of different mRNA isoforms, which can quantitatively affect protein synthesis, while leaving the encoded protein unchanged (Sun et al., 2012). APA may also involve events that occur in upstream regions (5′-UTRs, introns, or protein-coding regions; these isoforms are collectively called noncanonical in this report). Polyadenylation within the protein-coding region of the pre-mRNA will generate transcripts that lack a translation termination codon (non-stop mRNAs). In yeast (Saccharomyces cerevisiae) and mammals, non-stop RNAs are unstable and rapidly degraded through the non-stop decay mechanism (Frischmeyer et al., 2002; Vasudevan et al., 2002; Akimitsu et al., 2007). Polyadenylation within introns can result in alternative protein product synthesis or affect the stability of the mRNA through the nonsense-mediated mRNA decay (NMD) pathway (Hogg and Goff, 2010; Hwang and Maquat, 2011). Surprisingly, APA within 5′-UTRs also occurs, precluding transcription of the open reading frame.
In the past few years, studies have highlighted the important roles of APA in the control of global physiological events such as cellular proliferation, differentiation, transformation, and organ development (Lutz and Moreira, 2011). This is true for APA events that lead to the production of noncanonical mRNA isoforms, affecting the fate of the transcript and the nature of the products of translation. For example, in yeast, the induction of respiration-dependent growth leads to increases in the levels of several mRNA isoforms derived from polyadenylation within protein-coding regions (Sparks and Dieckmann, 1998). In genome-wide studies in yeast, environmental stresses were found to upregulate the levels of non-stop transcript isoforms generated by polyadenylation within protein-coding regions (Yoon and Brem, 2010). In Arabidopsis thaliana, intronic-APA regulates the balance of non/functional mRNA isoforms encoded by the FCA gene, a central flowering time regulator (Simpson et al., 2003; Duc et al., 2013) and by the gene that encodes RPP7, a receptor involved in defense responses (Tsuchiya and Eulgem, 2013). Using genome-scale approaches, numerous noncanonical mRNA isoforms with polyadenylated 3′ ends that lie within intronic, 5′-UTR, and protein-coding regions have been described in plants (Shen et al., 2011; Wu et al., 2011, 2014). In Arabidopsis, the set of genes that encode mRNAs with polyadenylation sites occurring within protein-coding regions and introns is enriched in those that are associated with stress responses and those that encode putative receptor proteins (Wu et al., 2011). In addition, there is a modest evolutionary conservation of APA events that lead to noncanonical mRNA isoforms in Arabidopsis and Medicago truncatula (Wu et al., 2014). These reports indicate that noncanonical polyadenylated mRNA isoforms are a standard component of the transcriptional output in plants.
We hypothesized that abiotic stress may lead to changes in polyadenylation site usage, leading to mRNA isoforms with differing stabilities or protein-coding capacities. Accordingly, we conducted a study to assess differential poly(A) site usage in Arabidopsis seedlings exposed to hypoxic conditions and to determine the relative stabilities and ribosome associations of mRNA isoforms derived from APA. We found that hypoxia increases the levels of mRNA isoforms with 3′ ends that lie in coding regions (non-stop mRNAs) and within introns. Interestingly, non-stop mRNAs and mRNA isoforms with 3′ ends that lie within introns were less stable than mRNAs with 3′ ends that lie within 3′-UTRs. These mRNA isoforms were less likely to be associated with ribosomes, especially in plants subjected to hypoxic conditions. A number of mRNA isoforms were identified that are generated by APA that have 3′ ends within annotated 5′-UTRs. Remarkably, these transcripts were present in polysomes in seedlings subject to normal conditions, but less under hypoxia. These findings indicate that the production of noncanonical mRNA isoforms is an important component of the response of plants to hypoxia. Moreover, they demonstrate that noncanonical mRNA isoforms have different properties and may be considered as three distinct classes of RNAs. Together, they indicate that production of noncanonical mRNA isoforms is a means by which transcriptional output is directed into decay pathways and is thus a global mechanism for negative gene regulation in plants.
General Experimental Approach for Studying Poly(A) Site Choice
An overview of the experimental approach used to evaluate polyadenylated RNA 3′ ends in Arabidopsis (Col-0) seedlings is summarized in Figure 1. Briefly, total or polysomal RNA was isolated from plants grown under control conditions, after hypoxic stress, or following treatment with the transcriptional inhibitor cordycepin. For each experimental treatment, samples were prepared in triplicate (biological replicates, pool of ∼30 whole plants per condition). Isolated RNA was used to prepare libraries of poly(A) tags (PATs), consisting of short cDNAs of 3′-terminal mRNA sequence primed with a fixed 18-nucleotide oligo(dT) tract. PAT libraries were sequenced on the Illumina platform, yielding 600,000 to 25 million individual sequence reads per library (Supplemental Data Sets 1A and 1B). Pooled libraries were demultiplexed using adaptor barcode sequences and aligned to the Arabidopsis genome.
Aligned reads were analyzed in several ways. First, they were used to assess overall gene expression by counting the total numbers of reads that map to each annotated Arabidopsis gene. The results were compared with similar published RNA-seq data to evaluate the consistency of the treatments. Second, poly(A) site analysis was conducted using a dedicated computational pipeline (Bell et al., 2016). For this, mapped reads were trimmed to a single nucleotide coordinate corresponding to the mRNA-poly(A) junction. These reduced tags were used to define poly(A) sites (PASs) and poly(A) site clusters (PACs), which are sets of individual sites that lie within 24 nucleotides of one another (Supplemental Data Sets 1C and 1D). The collection of experimentally determined PASs and PACs was used to determine the genomic distributions of PATs (Supplemental Data Set 1E) and to quantify relative poly(A) site usage in different biological replicates and experiments (Figure 1, middle and right panels, respectively).
Differential Poly(A) Site Choice in Plants Subjected to Hypoxia
To study hypoxia-induced processes, poly(A) site choice was studied in seedlings exposed to 2 h of hypoxic conditions, as well as in seedlings maintained under nonstress conditions (see Methods). Seedling growth and treatment conditions were identical to those used in prior experiments to monitor dynamics in total mRNA abundance, association with polysomes, and ribosome footprints (Juntawong et al., 2014a). PATs prepared from these plants were used to estimate fold change in transcript abundance in response to hypoxia; the results were compared with previously obtained total cellular poly(A)+ RNA data (Juntawong et al., 2014a) (Figure 2A). There was a good correlation between the data generated by RNA-seq and PAT-seq with samples produced at different times (correlation coefficient r1 = 0.707 and r2 = 0.912, using the whole transcriptome and only genes whose expression change by 2-fold or more, respectively). There were 777 gene transcripts that were significantly upregulated based on PAT counts (fold change > 2, false discovery rate [FDR]-corrected P value < 0.05) under hypoxic conditions; in this group, genes associated with responses to abiotic stresses were significantly overrepresented (Supplemental Data Sets 2A and 2B). Conversely, there were 1166 significantly downregulated gene transcripts based on PAT counts (fold change < −2, FDR-corrected P value < 0.05) in hypoxic conditions; genes associated with membrane functionality were significantly overrepresented in this set (Supplemental Data Sets 2A and 2B). These results corroborate previous reports (Mustroph et al., 2009b, 2014) and establish the utility of PATs for genome-wide studies.
To determine the effect of hypoxia on poly(A) site choice, individual PACs were evaluated for relative usage under control and hypoxic conditions. Thus, the number of tags that mapped to a given PAC was divided by the total number of tags that map to the associated gene, yielding a value for fractional usage of the PAC. The ratio of this value in hypoxic and control samples was used to evaluate changes in poly(A) site choice in response to hypoxia.
As stated above, poly(A) sites may lie at multiple locations within genic regions (5′-UTR, exon, intron, and 3′-UTR). As polyadenylation within functionally distinct regions may reflect different outcomes, the changes in relative usage of sites in each class in response to hypoxic stress were determined (Figure 2B). For reference, the changes in usage of all sites in the study were displayed as well ("Gene" in Figure 2B). For the collection of all sites, the median ratio of apparent usage in hypoxic plants compared with control plants was close to 0 (Figure 2B). A similar result was seen for sites that map to 3′-UTRs; however, the 25 to 75 percentile range for 3′-UTR-situated sites was much narrower than the same range for all sites, suggesting that most of the variability in poly(A) site choice induced by hypoxia was due to changes in sites that fall in other genic locations. This was indeed the case, as the apparent usages of sites that map to protein coding regions ("CDS" in Figure 2B), 5′-UTRs, and introns were greater in hypoxic plants (Figure 2B).
The increase in levels of mRNA isoforms with 3′ ends in coding sequences (CDSs), 5′-UTRs, and introns is also illustrated by consideration of the fraction of each mRNA isoform whose apparent usage changes by more than 2-fold after exposure to hypoxic conditions (Figure 2C). As seen in Figure 2B, 6% of all mRNAs with 3′ ends in CDSs were upregulated by at least 2-fold, as were more than 10% of all isoforms with 3′ ends in 5′-UTRs and introns, respectively. In contrast, roughly 0.3% of isoforms with 3′ ends in 3′-UTRs were upregulated. For all four classes, the numbers of downregulated isoforms were <4%.
Several individual examples were confirmed using a quantitative RT/PCR approach. For this, one primer set was designed that would query the combined expression of canonical and noncanonical isoforms for a given gene, while another set was designed that would query only canonical isoforms (the locations of amplification products are shown with purple and gray bars in Figures 3B to 3G). As shown in Figure 3A, in all cases there was a discrepancy in the relative changes in mRNA levels determined by the two sets of primers that matched the results seen in the high-throughput sequencing data (Figures 3B to 3G).
To further explore the consequences of APA on gene expression, the overall expression of genes subject to different modes of APA was assessed. For this, genes were classified according to their association with different modes of APA-possessing poly(A) sites that lie within 5′-UTRs, CDSs, or introns. All of these genes also possess poly(A) sites that lie within annotated 3′-UTRs, such that alternative poly(A) site choice involving the generation of canonical and noncanonical mRNAs is possible. Two additional classes of genes were assembled: those with multiple poly(A) sites that lie within more than one noncanonical location (e.g., a site in CDS and a site within an intron) and those with sites that fall only in 3′-UTRs (e.g., genes that do not encode noncanonical mRNA isoforms). For these classes of genes, the log2-fold changes in gene expression reported by Juntawong et al. (2014a) were retrieved and plotted, as shown in Figure 2E. The aggregate overall expression of genes in the different classes was somewhat lower in hypoxic plants, reflecting the genome-wide transcriptional responses noted by others (Mustroph et al., 2009b, 2014; Juntawong et al., 2014a). However, the downregulation was much more pronounced for genes with poly(A) sites that fall within the CDS. Interestingly, genes with sites that fall within 5′-UTRs and introns exhibited very little downregulation compared with the other classes of genes.
To further explore hypoxia-associated poly(A) site choice, those individual sites whose differential usage was the most prominent (FDR-adjusted P value < 0.05, determined using the DEX-seq package) were identified and evaluated. Over 569 sites satisfied this criterion; these sites were associated with 363 genes (Supplemental Data Set 3A). Genes associated with defense and stress responses were significantly overrepresented in this set of 363 genes (Figure 2D, bootstrap > 1.5, P value < 0.01; Supplemental Data Set 3B); in particular, genes encoding putative receptors tended to be affected in hypoxic plants. For these genes, there was a consistent increase in the levels of mRNA isoforms derived from polyadenylation within the CDS; examples are shown in Supplemental Figure 1. Analysis of this set of genes using MapMan indicated an interesting effect of APA on the expression of genes associated with nitrogen metabolism and transport processes. Specifically, the two Arabidopsis genes that encode nitrate reductase (NIA1 and NIA2) showed an unusual hypoxia-induced usage of poly(A) sites that lie within the third and first intron of each gene, respectively (Supplemental Figure 2).
Differential Stabilities of mRNA Isoforms
As stated above, some classes of noncanonical mRNA isoforms are expected to be subject to mRNA surveillance mechanisms. To shed light into this, the relative stabilities of the different classes of mRNA isoforms were determined. For this, the approach taken by Gutierrez et al. (2002) was used. Two-week-old Arabidopsis seedlings were treated with 200 µM cordycepin for 120 min to block nuclear transcription. RNA was isolated and used for PAT-seq library preparation and analyzed as described in the preceding sections.
To confirm that the cordycepin treatment was effective, an analysis of overall steady state mRNA levels was conducted, and the results were compared with those of Gutierrez et al. (2002) and Kim and Chen (2011). For this, relative expression levels were determined using the numbers of PATs that map to individual annotated genes. The rationale for this analysis is that, while most mRNAs will decay within 120 min, the decay of the most unstable mRNAs will be more rapid and will thus stand out when relative expression levels are calculated. This analysis is summarized in Supplemental Data Set 2A. A total of 2378 unstable transcripts could be identified (transcripts that decline in abundance by 2-fold or more within 120 min after the addition of cordycepin). In agreement with the previous studies, the set of genes that encode relatively unstable mRNAs tended to be enriched for transcription factors and responses to hormone stimulus, abiotic and biotic stress, and light stimulus and radiation (Supplemental Data Set 2C). Moreover, there was good agreement between the list of genes encoding unstable mRNAs in this study and those published previously (Supplemental Data Set 2D); 83.2% of the unstable transcripts identified by Gutierrez et al. (2002) and 79.1% of the unstable transcripts identified as unstable in the study by Kim and Chen (2011) were also flagged as unstable here. These results indicate that the cordycepin treatment was effective, and by coupling this inhibitor with the use of PAT libraries to discern the frequency of poly(A) site usage, it should be possible to monitor the relative stabilities of different mRNA isoforms.
Therefore, the relative stabilities of individual mRNA isoforms were assessed. As was done for the study in Figure 2, the relative usage of each poly(A) site was calculated for the t = 0 (C0) and t = 120 (C120) samples. The ratios of usage in the C0 and C120 samples were determined and log2 transformed. In this analysis, values less than zero indicate lower stabilities (and, hence, a lower contribution of the respective site to the total steady state mRNA level), while values greater than zero suggest a greater stability of the associated mRNA isoform. The values for each class of RNA isoform were then displayed in a box plot (Figure 4A). For all sites taken together, the median value was 0.31. A similar result was obtained for all sites that fell within 3′-UTRs (0.38). Sites that fell within annotated 5′-UTRs had a slightly lower median value (0.36), albeit one that was not significantly different from the median obtained with 3′-UTR sites. In contrast, mRNA isoforms that end within protein-coding regions had a significantly lower value (−0.4). Intronic sites had still lower values (−0.58).
The lesser stability of mRNA isoforms with 3′ ends in CDSs and introns is also illustrated when considering the fraction of each mRNA isoform that falls into "unstable" and "stable" categories. If these categories are defined as isoforms whose fractional contribution to all PATs associated with a given gene changes by more than 2-fold, then more than 25% of all non-stop mRNA isoforms (polyadenylated within the CDS and thus lacking a stop codon) fall into the unstable category, while 40% of all isoforms with 3′ ends within introns are unstable (Figure 4B). In contrast, <14 and 2% of mRNA isoforms with 3′ ends in 5′-UTRs or 3′-UTRs, respectively, were unstable, while more than 18 and 8% for each category could be classified as stable, respectively. Some representative examples of unstable mRNA isoforms are shown in Figure 4C; these examples illustrate the general outcome of the assay, showing the disproportionate decrease in some isoforms compared with others. These results further illustrate that mRNAs with 3′ ends within CDSs and introns are, in aggregate, less stable than other mRNA isoforms under standard growth conditions. Analysis by RT-qPCR confirmed these examples (Figure 4D). Altogether, these results indicate that mRNA isoforms derived from polyadenylation within protein-coding regions and introns are less stable than other isoforms.
Differential Translation of mRNA Isoforms upon Hypoxia
The preceding results indicate that hypoxic conditions lead to increases in the steady state levels of truncated mRNA isoforms derived from proximal poly(A) site choice. These isoforms should not encode full-length polypeptides, raising the question as to whether these isoforms are actively translated. To study this, the poly(A) site profiles were determined for mRNAs associated with polysomes isolated by immunopurification via an epitope-tagged protein of the 60S ribosomal subunit. For this, RNA was purified from polysomes of control and hypoxic plants and PAT libraries were constructed. As before, an analysis geared toward the determination of overall "expression" level was conducted using PATs as the proxy for mRNA transcript. The relative levels of PATs in the total RNA samples were then compared with those from polysomal RNA, and the relative enrichment or depletion of the products of particular genes in the polysomal samples was determined (Supplemental Data Sets 2A, 2E, and 2F). These results were compared with those of previous studies; as seen in Figures 5A and 5B (for normoxia and hypoxia, respectively), there was good agreement with earlier results (Juntawong et al., 2014a).
Subsequently, the relative abundances of individual mRNA isoforms in RNA isolated from polysomes was calculated and compared with those seen in total RNA to determine if isoforms or classes were more or less prevalent in purified polysomes than in total RNA. In control plants (Figure 5C), the median value for polysome enrichment was close to 0 ("Gene" in Figure 5C). A similar result was seen for mRNA isoforms with 3′ ends that map to 3′-UTRs and introns. The median polysome enrichment for isoforms with 3′ ends that map to protein-coding regions was lower, whereas the enrichment for mRNAs with 3′ ends in 5′-UTRs was higher (Figure 5C). Almost 10% of the individual CDS-situated sites were significantly underrepresented, while ∼5% were overrepresented (Figure 5E, left side). Similar fractions of intron-derived isoforms under- and overrepresented in polysomes were apparent, while there were many more overrepresented 5′-UTR-derived isoforms than underrepresented isoforms (around 25 and 5%, respectively). In contrast, more than 95% of isoforms with 3′ ends in annotated 3′-UTRs were neither over- nor underrepresented in polysomes.
In hypoxic plants, the median value for polysome enrichment was slightly greater than 0 when the entire set of mRNA isoforms was examined ("Gene" in Figure 5D). A similar result was seen for mRNA isoforms with 3′ ends that map to 3′-UTRs. The median polysome enrichments for isoforms with 3′ ends in protein coding regions and introns were lower than the ratios for isoforms with 3′ ends that lie within 3′-UTRs (Figure 5D). Almost 15% of the CDS isoforms were underrepresented in polysomes, while <6% were overrepresented (Figure 5E, right side).
Remarkably, for mRNAs with 3′ ends in 5′-UTRs, the median polysome enrichment in hypoxic plants was much lower than was seen for these isoforms in control plants (compared with "5'-UTR" in Figures 5C and 5D). For these isoforms, almost 30% were underrepresented in polysomes in hypoxic plants (Figure 5E, right side).
To further explore polysome-associated APA under normal and hypoxic conditions, those individual sites whose usages in polysomes and total RNA were significantly different (FDR-adjusted P value < 0.05, determined using the DEX-seq package) were identified and evaluated. A total of 278 sites from 189 genes satisfied these criteria in normal plants, as did 1075 sites involving 684 genes in hypoxic plants (Supplemental Data Sets 3C and 3D). Genes associated with photosynthesis, ribosomes, and stress responses were significantly overrepresented in both sets of genes (Figure 5F, bootstrap > 1.5, P value < 0.01; Supplemental Data Set 3E). Genes associated with intermediary metabolism, including that of hormones, were overrepresented in the list of genes affected by hypoxia-induced changes in polysome association. These results indicate that the production of noncanonical mRNAs seems to have a disparate influence, through both the shunting of transcriptional output into the production of noncanonical mRNAs as well as the diminished translation of these mRNA isoforms, on the functioning of genes that are involved in many aspects of intermediary metabolism and growth.
The association of 5′-UTR isoforms with polysomes is unexpected, as these isoforms do not seem to be particularly enriched for so-called upstream open reading frames; only ∼25% of these isoforms have open reading frames of 25 codons or longer that are defined by AUG translation initiation codons and an additional 10% have open reading frames defined by CUG initiation codons. Inspection of ribosome footprinting data (Juntawong et al., 2014a) corroborates this association (Figure 6). For example, a distinct ribosome footprint (in the TRAP_RS) tracks in Figure 6 can be discerned in the At2g47240 and At3g23030 genes, which encode polysome-associated mRNA isoforms derived from polyadenylation within 5′-UTRs (Figures 6A and 6B, respectively). Such footprints are not seen with other genes (such as At5g46780 and At3g30300) that do not encode 5′-UTR isoforms (Figures 6C and 6D). Thus, the association that can be inferred from the results shown in Figure 5 is authentic. The shifts in this association in hypoxic plants suggests the presence of an additional, hitherto unknown, regulatory response involving 5′-UTR mRNA isoforms, perhaps representing an additional contribution that APA makes to stress-related gene regulation.
Distinctions between Different Classes of Noncanonical mRNA Isoforms
In plants, as in other eukaryotes, alternative polyadenylation is a widespread and pervasive process, potentially affecting a large majority of genes for a given species (Shen et al., 2011; Wu et al., 2011, 2014; Sherstnev et al., 2012). In plants, a majority of genes that possess multiple poly(A) sites have at least one site situated upstream of the 3′-UTR, proximal to the associated promoter (Shen et al., 2011; Wu et al., 2011, 2014; Fu et al., 2016). The results presented in this study indicate that the products of proximal poly(A) site choice (collectively, noncanonical mRNA isoforms) may be differentiated according to the location of the poly(A) site within the transcriptional unit.
mRNAs with 3′ Ends That Lie within Protein-Coding Regions
One class of noncanonical mRNA isoform consists of mRNAs with 3′ ends that lie within protein-coding regions. The mRNA isoforms derived from polyadenylation within CDSs will, with few exceptions, lack translation termination codons. In animals and yeast, such RNAs (termed non-stop RNAs) are less stable than canonical mRNAs (Frischmeyer et al., 2002; van Hoof et al., 2002). The decay of non-stop RNAs is mediated by ribosome-associated factors that act to recognize stalled ribosomes and recruit the exosome so as to facilitate degradation of the associated RNA (Klauer and van Hoof, 2012). The results presented in this study confirm that mRNA isoforms derived from polyadenylation within protein-coding regions are generally less stable than other isoforms (Figure 4) and are thus consistent with the presence of a quality control process in plants analogous to the non-stop decay pathway.
In hypoxic conditions, the relative levels of putative non-stop mRNAs in total RNA increase (Figure 2). In addition, in both normoxic and hypoxic conditions, these mRNA isoforms are somewhat underrepresented in polysome-associated RNA populations (Figure 5). This latter consideration, along with the lower stability of these mRNA isoforms, indicates that one aspect of the response of plants to hypoxia is a general redirection, via the process of APA, of transcriptional output into nonproductive outcomes or pathways. In addition to the dynamics of this class of noncanonical mRNA, this redirection has an effect on the overall levels of canonical, functional mRNA isoforms; this is revealed by the decreased levels of functional mRNA isoforms encoded by genes that are affected by APA within protein-coding regions (Figure 2E).
mRNAs with 3′ Ends That Lie within Annotated Introns
Several instances of alternative polyadenylation within introns have been described in plants (Giranton et al., 1995; Bassett et al., 2002; Tang et al., 2002; Simpson et al., 2003; Duc et al., 2013; Tsuchiya and Eulgem, 2013). The distal/proximal choice of poly(A) sites in pre-mRNAs encoded by the gene that encodes lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase in cotton (Gossypium hirsutum) determine whether LKR or a bifunctional LKR/saccharopine dehydrogenase polypeptide is produced (Tang et al., 2002). Proximal poly(A) site choice in pre-mRNAs encoded by the Arabidopsis FCA gene leads to the production of a truncated mRNA (with a 3′ end lying within a large intron) and seems to play a negative regulatory role in the expression of this gene (Simpson et al., 2003). A similar scenario and negative regulatory role are operative for the Arabidopsis FPA gene (Duc et al., 2013). Some genes encoding receptors involved in ethylene signaling (Bassett et al., 2002), self-incompatibility (Giranton et al., 1995), and defense signaling (Tsuchiya and Eulgem, 2013) are affected by proximal poly(A) site choice, with proximal polyadenylation seeming to yield truncated mRNAs that may possibly encode shorter polypeptides with some regulatory function. In this study, among genes affected by hypoxia-induced APA within introns are the two Arabidopsis genes that encode nitrate reductase (NIA1 and NIA2); as is the case with other instances listed in the preceding examples, hypoxia induces the production of NIA1/2-derived transcripts that may encode truncated polypeptides.
In all of these cases, the contributions that intronic polyadenylation make to gene regulation have been suggested to involve the production of truncated polypeptides, which may have biochemical activities or may serve as a nonproductive outlet for the transcriptional output of the gene. The results presented here indicate that mRNA isoforms derived from polyadenylation within introns are collectively less stable than canonical mRNA isoforms (Figure 4). This suggests that, as proposed for APA within CDSs, intronic APA may represent a mode of negative regulation that redirects transcriptional output toward mRNAs with lower stability. However, in contrast to what is seen with putative non-stop mRNAs, intronic APA is not associated with a marked decrease in the levels of gene expression (Figure 2E). This suggests that intronic APA is not merely a mechanism for shunting transcriptional output into RNA quality control pathways at the expense of the production of functional mRNAs. Rather, intronic APA may provide a mechanism for generating novel, truncated polypeptides of particular function, and the general lower stability of this class of mRNA isoforms may reflect other aspects of the structures of these isoforms.
The reasons for the reduced stability of mRNA isoforms derived from intronic APA are not entirely clear. As opposed to RNA isoforms derived from included (retained) introns (a common mode of alternative splicing in plants), isoforms with 3′ ends that lie within introns should not have premature translation termination codons in the usual sense (this sense being that they would be followed by an unusually long 3′-UTR with one or more downstream splice junctions). While introns in Arabidopsis that possess poly(A) sites are longer than the typical intron [the average length of introns with poly(A) sites being 270 nucleotides; Wu et al., 2011], they are not so long that polyadenylation within them would lead to unusually long 3′-UTRs (a determinant of NMD; Hogg and Goff, 2010). Specifically, the average 3′-UTR length in the latest version of the TAIR10 Arabidopsis database is 237 nucleotides, whereas the triggering of NMD is usually associated with 3′-UTR lengths of greater than 350 nucleotides (Kalyna et al., 2012). Transcripts derived from intronic polyadenylation are not non-stop RNAs, as they invariably possess one or more in-frame stop codon in the intron-derived part of the 3′-UTR. Other, as yet unidentified, mechanisms that reflect interplay between splicing, polyadenylation, and mRNA turnover may be in force in determining the reduced stability of these mRNA isoforms.
mRNAs with 3′ Ends That Lie within Annotated 5′-UTRs
Perhaps the most enigmatic of the classes of noncanonical mRNA isoforms described in this study are those derived from polyadenylation within 5′-UTRs. The levels of these isoforms increase dramatically in response to hypoxia (Figure 2). However, these isoforms differ little in their stability from isoforms produced by canonical poly(A) site choice (Figure 4). Another surprising feature of mRNAs with 5′-UTR-situated poly(A) sites is the association of some of these RNAs with polysomes in nonstressed conditions (Figure 5). As stated above, these isoforms do not seem to be particularly enriched for annotated upstream open reading frames, and most lack open reading frames of more than 25 codons; there thus seems to be little context by which translation might initiate on these mRNAs. Of all classes of noncanonical mRNAs, the translation of this class is most affected by hypoxia; thus, in normoxic plants, 5′-UTR-derived isoforms are somewhat enriched in polysomes, while in hypoxic plants, they are substantially underrepresented in the set of polysome-associated mRNAs (Figure 5). These characteristics distinguish this class of noncanonical mRNA from other noncanonical mRNA isoforms and raise the possibility that they constitute an as yet unrecognized class of RNA.
While the exact means by which these novel mRNA isoforms are generated are not yet known, one set of results presented here argues against one possible origin. This model holds that these isoforms may be formed by polyadenylation of prematurely terminated Pol II transcripts. The surveillance of premature termination products is linked with a different mode of polyadenylation followed by nuclear exosome-mediated degradation and yields relatively unstable products (Preker et al., 2008; Fox and Mosley, 2016). The observation that Arabidopsis mRNA isoforms derived from polyadenylation within 5′-UTRs are not significantly less stable than canonical mRNA isoforms is not consistent with the idea that the truncated mRNAs are produced by coupled termination and nuclear surveillance.
Other considerations may also be informative as far as the origins and functions of these isoforms. The nature of these isoforms—their enrichment in polysomes in control plants, their relative stabilities compared with other noncanonical isoforms as well as with canonical mRNAs, and the dramatic changes in abundance and polysome association in hypoxic plants—does not easily lend itself to precedent-based interpretations. On one hand, translation of these mRNAs may be fortuitous because of the presumed presence of a 5′-cap as well as a poly(A) tail. However, there is at least a cursory similarity to the identification of ribosome-associated long noncoding RNAs seen in zebra fish. Specifically, in zebra fish, ribosome footprints on long noncoding RNAs resembles the distribution of ribosomes seen along 5′-leader sequences of canonical mRNAs (Chew et al., 2013). Arabidopsis mRNA isoforms with 3′ ends that lie within 5′-UTRs are analogous to mRNA leaders, albeit without significant protein-coding regions. Accordingly, it is possible that these short mRNA isoforms are in fact a subclass of long noncoding RNAs. This possibility is exciting, as it raises the prospect that these mRNA isoforms may play regulatory roles similar to those ascribed to long noncoding RNAs.
Alternative Polyadenylation and the Responses of Plants to Hypoxia
In plants subjected to hypoxic conditions, the abundances of mRNA isoforms derived from polyadenylation at the various proximal sites (5′-UTRs, CDSs, and introns) all increase (Figure 2). The levels of non-stop RNA isoforms increase modestly (albeit significantly), while isoforms with 3′ ends within 5′-UTRs and introns increase substantially. The mechanisms that underlie these increases have not been studied, but other work and considerations that flow from this study support some mechanisms above others. In a general sense, these increases in abundance may be due to increased usage of the respective poly(A) sites, such that more of the transcriptional output is directed toward proximal polyadenylation. Several of the activities of individual polyadenylation complex subunits are affected by phosphorylation, methylation, calmodulin, disulfide remodeling, and ubiquitin (Jacob and Rose, 1984; Thuresson et al., 1994; Colgan et al., 1996; Mizrahi and Moore, 2000; Mouland et al., 2002; Delaney et al., 2006; Ryan, 2007; Addepalli and Hunt, 2008; Ryan and Bauer, 2008; Addepalli et al., 2010; Martin et al., 2010). In addition, alterations in the activities of individual poly(A) complex subunits are often accompanied by large-scale shifts in poly(A) site choice (Jenal et al., 2012; Martin et al., 2012; Thomas et al., 2012; Duc et al., 2013; Li et al., 2015). Hypoxia is associated with transient activation of MPKs and spikes in cytosolic calcium as well as dynamics in reactive oxygen species, along with a reduction in ATP availability and a shift to anaerobic metabolism that can alter cytosolic pH (Chang et al., 2012). All of these signaling events have the potential to alter various subunits of the polyadenylation complex and thus trigger shifts in poly(A) site choice.
Alternatively, the increases in the levels of noncanonical mRNA isoforms in hypoxia may reflect, not increased usage of the associated poly(A) sites, but an increased stability of the various isoforms. For example, the reduction of global protein synthesis by ∼50%, accompanied by the sequestration of mRNAs into cytosolic granules containing Oligouridylate Binding Protein 1C (Sorenson and Bailey-Serres, 2014), may limit the degradation of noncanonical transcripts. The association of RNA quality control processes (in particular, nonsense-mediated decay and non-stop decay) with ribosomes (Simms et al. 2017) and the decreased association of noncanonical mRNA isoforms with polysomes in hypoxic plants (Figure 5) may also contribute to an increase in the levels of noncanonical transcripts. Of course, this assumes that the decay of various noncanonical mRNA isoforms is mediated by these established pathways. For isoforms with 3′ ends that fall within protein coding regions, this seems likely. However, for the other classes of noncanonical mRNAs, this is not as clear. As discussed above, intron-derived isoforms do not have properties consistent with NMD substrates, and 5′-UTR-derived isoforms likewise lack features that are associated with RNA quality control. These caveats notwithstanding, it is probable that alterations in RNA turnover rates contribute to some of the alterations in the levels of noncanonical transcripts in hypoxic plants.
It has been proposed that the production of different mRNAs due to alternative RNA splicing contributes to a fine-tuning of the expression of the associated genes, by shunting transcriptional output into NMD pathways (Green et al., 2003; Lewis et al., 2003; Lareau et al., 2007; Palusa and Reddy, 2010; Kalyna et al., 2012; Drechsel et al., 2013). The results presented here suggest a similar role for APA in plants, especially for events that lead to polyadenylation within protein-coding regions. In plants, genes that encode NBS-LRR (nucleotide binding site-leucine-rich repeat) receptors are subject to APA (Tan et al., 2007), and genes in this set are often affected by polyadenylation within protein coding regions (Thomas et al., 2012; Wu et al., 2014). Many such genes are among those affected by APA in response to hypoxia (Figure 2D). While these proteins are typically associated with innate immunity and defense responses (Marone et al., 2013), there are reports of the involvement of particular NBS-containing receptors with responses to abiotic stress (Huang et al., 2010; Zbierzak et al., 2013; Sarazin et al., 2015). More generally, there is considerable overlap in the molecular responses of plants to biotic and abiotic stress. For example, submergence enhances innate immunity mediated by the transcription factor WKRY22 in Arabidopsis (Hsu et al., 2013). Genes that encode receptors require precise control, and misregulation of these can have detrimental consequences; this is especially true in instances where such genes are overexpressed (Kim et al., 2010; Kato et al., 2014). The results presented here suggest that negative regulation via APA, manifest as indicated in Figure 2E, contributes to the fine-tuning of the expression of genes associated with stress and thus contributes to the global responses of plants to hypoxia.
An additional functional category identified in the meta-analysis as being associated with hypoxia-related APA relates to nitrogen metabolism (MapMan analysis summarized in Figure 2D). This category is flagged largely because of APA involving the two nitrate reductases (NIA1 and NIA2; Supplemental Figure 2). In both instances, poly(A) sites located within introns are utilized in hypoxic plants but not in controls, leading to the production of truncated mRNAs capable of encoding polypeptides consisting of the molybdenum cofactor binding domain, a part of the enzyme capable of generating nitrite in vitro when provided with an external electron transport capacity (Fischer et al., 2005). Nitric oxide enhances the turnover of the key ethylene-responsive transcription factors (ERF-VIIs) that mediate low-oxygen sensing and upregulate the core low oxygen response in hypoxic cells (Gibbs et al., 2011, 2014). Moreover, the nia1nia2 mutant is defective in the ERF-VII and hypoxia mediated positional cue that regulates apical hook opening (Abbas et al., 2015). Nitrate reductase genes are often upregulated under conditions of low oxygen (Mustroph et al., 2010; Juntawong et al., 2014b), and nitrate supplementation alters the physiological responses of plants to low oxygen (Allègre et al., 2004). The induction of the ability to generate truncated nitrate reductase-related polypeptides via APA (Supplemental Figure 2) is yet another aspect of the response of nitrate reductase genes in low oxygen. The significance of this facet of nitrate reductase gene expression is not clear at the moment. Truncated polypeptides may interact with and in some way modify the activities of the full-length enzymes. The truncated polypeptides may retain the nitrite-generating activity in vivo that is seen in vitro (Fischer et al., 2005). This may provide plants with a means by which some nitrate reductase activity can be linked with alternative electron transport capacities under conditions of low oxygen (Bailey-Serres and Voesenek, 2008). Alternatively, these truncated polypeptides may contribute (along with the plant cysteine oxidases) to the rapid oxidation of the N-terminal cysteine of the ERF-VIIs upon re-aeration, which is required to initiate their turnover by the N-end rule pathway (Weits et al., 2014; Voesenek and Bailey-Serres, 2015). The tight control of ERF-VII localization, abundance, and function limits their activation of the transcription of genes encoding enzymes necessary for anaerobic metabolism, which is energetically expensive when oxidative phosphorylation is feasible but a necessity for the maintenance of cell viability during a transient limitation in oxygen (Bailey-Serres and Voesenek, 2008; Bailey-Serres et al., 2012a).
The results presented here reveal a broad-ranging reach of alternative polyadenylation in the response of Arabidopsis to hypoxia. APA generates three distinct classes of noncanonical mRNA, distinguishable by their inherent stabilities, associations with polysomes, and responsiveness to hypoxia. Many of the products of APA are relatively unstable, and their levels and associations with polysomes change substantially under conditions of low oxygen. An important contribution that APA makes to gene regulation in hypoxic plants involves the shunting of transcriptional outputs to nonproductive outcomes; thus, as is seen with alternative splicing, APA constitutes a mode of negative regulation. In the context of the response of plants to hypoxia, this mode of regulation especially affects genes that encode stress-related receptors. Additionally, APA affects the expression of nitrate reductase genes in interesting ways, suggesting that APA plays a role in mediating the links between low oxygen and nitrate signaling or metabolism in plants.
Plant Material and Treatment
Arabidopsis thaliana ecotype Col-0 wild-type and transgenic plants expressing NH2-His6-FLAG epitope-tagged ribosomal protein L18 via a near-constitutive promoter (35Spro:FLAG-RPL18) in Col-0 (Zanetti et al., 2005; Mustroph et al., 2009b; Sorenson and Bailey-Serres, 2014) were used. For mRNA polyadenylation site analyses, 35Spro:FLAG-RPL18 seeds were surface sterilized, incubated at 4°C for 2 d, and grown on vertically positioned plates containing solid Murashige and Skoog medium (0.43% Murashige and Skoog salts, pH 5.75, and 29 mM [1% w/v] sucrose) in a growth chamber under long-day conditions (16-h light [∼120 µE s–1 m–2]/8-h dark photoperiod, the bulbs used were Sylvania Octron Eco Fluorescent) at 23°C (Sorenson and Bailey-Serres, 2014). Hypoxic stress was imposed after the end of the light period after 7 d growth by gassing in chambers with humidified 99.99% (v/v) argon for 2 h at 5 to 7 µmol photons m−2 s−1 light; control samples were maintained under the same conditions in chambers open to air as described (Sorenson and Bailey-Serres 2014).
For stability studies, 2-weeks-old Col-0 wild-type plants grown in Murashige and Skoog medium were transferred to a flask with incubation buffer (15 mM sucrose, 1 mM KCl, 1 mM PIPES, and 1 mM sodium citrate, pH 6.5). After a 30-min incubation (time 0), 3′-deoxyadenosine (cordycepin) was added to a final concentration of 200 µM. Tissue samples were harvested after 120 min and quickly frozen in liquid nitrogen (Johnson et al., 2000). Each sample (biological replicate) was the result of pooling ∼30 plants per condition.
In all cases, three biological samples and three independent experiments were performed.
Immunopurification of Polysomes
The immunopurification of ribonucleoprotein complexes from transgenic Arabidopsis line 35Spro:FLAG-RPL18 exposed to hypoxia and control conditions was accomplished as described (Mustroph et al., 2009a).
Poly(A) Tag Library Preparation and Sequencing
Total or polysomal RNA was isolated from Arabidopsis plants after treatment (control, hypoxia, and cordycepin) using the Trizol reagent and RNeasy columns (Qiagen). Quantity and quality measurements were taken using a NanoDrop spectrophotometer. Arabidopsis poly(A) tags (PATs) were generated with 1 µg of total RNA or 0.5 µg of polysomal RNA using Method B1 as described (Ma et al., 2014). The quality of the PAT library was checked with a Bioanalyzer using Agilent High Sensitivity DNA chips (Agilent Technologies), and concentrations were measured with a Qubit fluorometer and a Qubit dsDNA HS assay kit (Life Technologies). The resulting poly(A) tags were sequenced on an Illumina high-throughput sequencing DNA platform. In all cases, three independent biological replicates (pool of ∼30 whole plants) were used.
Poly(A) Tag and Gene Expression Analysis
The sequencing data from PAT-seq were processed using the pipeline as detailed previously (Bell et al., 2016). Briefly, using the CLC Genomics Workbench suite of tools (Qiagen Bioinformatics), the initial sequences obtained were demultiplexed and trimmed to remove the oligo(dT) tracts and sequencing adapters. The processed tags were then mapped to the Arabidopsis reference genome (TAIR10; Berardini et al., 2015) using the current version of CLC Genomics Workbench [individual mapping to evaluate the quality and determine gene expression and pooled sequences to carry out the poly(A) analysis]. The BEDTools suite was used to count PATs that mapped to individual annotated genes and to derive lists of individual PASs and PACs. Only sites and clusters defined by at least 10 individual PATs were retained for subsequent analyses. PAT, PAS, and PAC information was used to carry out three different analyses: (1) to determine PATs, PAS, and PAC distributions on annotated gene regions; e.g., 3′-UTR, 5′-UTR, intron, AMB, and protein-coding regions (AMB from AMBiguous are regions that have different annotations in different transcripts encoded by the same gene); (2) to assess statistically significant differences in poly(A) site usage in a gene-by-gene analysis using the DEX-seq package (Anders et al. 2012); and (3) to determine gene expression using the empirical analysis of DGE (digital gene expression) algorithm in CLC Genomics Workbench. For DGE, genes with a total filter cutoff of 2-fold change and FDR-corrected P values < 0.05 were selected as statistically significant.
A number of ancillary studies were conducted to assess the quality and reproducibility of the libraries prepared and analyzed in this study. These studies are described in the Supplemental Methods and shown in Supplemental Figures 3 to 7.
Quantitative RT-PCR
cDNA was produced with SMARTScribe reverse transcriptase (Clontech), using as starting material 1 to 2 µg of total RNA from three biological replicates. The fragments of interest were amplified by RT-qPCR using specific primers (P1, close to the 3′-UTR; P2, close to the APA-upstream region; and P3, located in APA-5′-UTR upstream region) and SsoAdvanced SYBR Green Supermix (Bio-Rad) as fluorescence dye. ACTIN2 was used as a housekeeping gene to normalize the expression of the genes investigated. The primers used are listed in Supplemental Table 1.
Statistical Analysis
Statistical treatment of the gene expression comparisons used the "exact test" to calculate the P value, assuming that the count data follows a negative binomial distribution (Robinson and Smyth 2008); thus, genes with FDR-corrected P values < 0.05 were selected as statistically significant. The usages of different classes of poly(A) sites were tested for significant differences using a hypergeometric probability test (Supplemental Data Set 4). The statistical method used by DEX-seq was as described (Anders et al. 2012), and an adjusted P value < 0.05 was considered to indicate statistical significance.
Functional Analysis
To search for ontology term enrichment in the different APA-associated gene classes, the classification SuperViewer Tool from Bio-Analytic Resource for plant biology (BAR Arabidopsis website, http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) was used. Significance threshold of P value < 0.01 and bootstrap > 1.5 were chosen as statistically significant for Gene Ontology and MapMan classification. The ontology term enrichment in differentially expressed genes was also analyzed using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/home.jsp). Significance threshold of P value < 0.01 and fold enrichment > 1.5 were chosen as statistically significant.
Accession Numbers
The raw data for this study may be found at NCBI SRA (https://submit.ncbi.nlm.nih.gov/subs/sra/) under SRA accession number SRP089899. PASs and PACs that are defined by the poly(A) tags are reported in Supplemental Data Sets 1D and 1E, respectively. The poly(A) metric output for the individual biological replicates comparison using PATapp are reported in Supplemental Data Sets 5A to 5H, and the poly(A) metric output for the comparison with previously published data is included in Supplemental Data Sets 5I and 5J. PACs from 3′ end using RNA-seq that overlap with PACs identify using PAT-seq are reported in Supplemental Data Set 6.
Supplemental Data
Supplemental Figure 1. Examples of hypoxia-induced APA involving genes that encode defense-associated receptors.
Supplemental Figure 2. APA involving the two Arabidopsis genes that encode nitrate reductase.
Supplemental Figure 3. Box plots of the pairwise poly(A) metric comparison between replicates of each sample data set.
Supplemental Figure 4. Box plots of the pairwise comparison of wild-type data sets.
Supplemental Figure 5. Confirmation of poly(A) sites using 3′-RACE.
Supplemental Figure 6. Comparisons of PAT-seq and RNA-seq coverage for poly(A) sites.
Supplemental Figure 7. S1 nuclease protection assay to map the 5′ end of the At1g03610 transcript.
Supplemental Table 1. Summary of oligonucleotides used in this study (5′–3′).
Supplemental Methods. Assessment of libraries and poly(A) site determinations.
Supplemental Data Set 1. Summaries of read mapping outcomes for the different libraries.
Supplemental Data Set 2. Gene expression analysis under different treatments in Arabidopsis thaliana.
Supplemental Data Set 3. DEX-seq analysis output.
Supplemental Data Set 4. Significance of poly(A) cluster usage in different isoforms.
Supplemental Data Set 5. Raw outputs of the analysis presented in Supplemental Figures 3 and 4.
Supplemental Data Set 6. PACs confirmed by RNA-seq.
We thank Carol Von Lanken for technical assistance. This work was supported by the National Science Foundation (MCB-1243849 to A.G.H. and MCB-1021969 to J.B.-S.).
All four authors contributed to the conceptualization and design of this study. L.d.L. performed the mRNA stability study and carried out the high-throughput sequencing, analyses, and validation experiments. R.S. performed the hypoxia treatment, polysome purification, and RNA extraction from hypoxic plants and polysomes. A.G.H. helped with the computational and statistical of sequencing data. J.B.-S also contributed to the analysis and presentation of sequencing data. L.d.L. and A.G.H. wrote the manuscript with critical input from R.S. and J.B.-S.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Arthur G. Hunt (aghunt00{at}uky.edu).
↵1 Current address: Department of Biology, University of Utah, Salt Lake City, UT 84112-6517.
↵[OPEN] Articles can be viewed without a subscription.
- alternative polyadenylation
- untranslated region
- nonsense-mediated mRNA decay
- poly(A) tag
- poly(A) site
- poly(A) site cluster
- false discovery rate
- coding sequence
- lysine-ketoglutarate reductase
- Received September 26, 2016.
- Revised May 17, 2017.
- Accepted May 30, 2017.
- Published May 30, 2017.


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We are running Server 10.3.1. Our router and firewall was reset over the weekend and our website went down. We redirected port 80 to port 6080 (the ArcGIS Server) and our data and maps are viewable on the website again. We can see the REST services directory from the web on http://gis.ourwebsite.com/arcgis/rest. The problem is, none of the other files or directories in our wwwroot folder (which contains the arcgis web adapter) are accessible from that URL. For example, http://gis.ourwebsite.com/file.ext returns a 404 error. We have hyperlinks to these files on our website.
I've tried enabling directory browsing on our default website directories in IIS 8.5, but this hasn't helped. Every folder underneath "arcgis" in this screenshot is inaccessible from the web.

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In 2020 the"VIII World Conference Violence in Schools will be held in the city of
Puerto Vallarta, Jalisco, México.
Stretching around the sparkling blue Bahía de Banderas and backed by lush palm-covered mountains, Puerto Vallarta (or just 'Vallarta' to many) is one of Mexico's most enticing coastal destinations. Each year millions come to laze on the dazzling sandy beaches, browse in the quirky shops, nosh in the stylish restaurants and wander the picturesque central streets and enticing malecón (boardwalk). There are activities aplenty, including boat trips, horseback rides, diving trips and day trips to the interior. After sunset, Vallarta takes on a new identity with pumping nightlife along the cobblestone streets.
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Room plan with breakfast
Price per person per night
Rates quoted in US dollars
To book click on the image of Sheraton or on the reservations button.
Sheraton Buganvilias Resort & Convention Center
Luxury room with ocean view
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Taxes and tips for breakfast buffet
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Rates will apply for up to five nights, in advance arrivals and departures after the VIII CMVE, according to availability at the hotel
Rates apply for a minimum of two nights stay
The plan does not include the Starbucks DELI
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0% VAT TAX LAW for international meetings in Mexico:
According to Mexican laws, the groups with sessions that take place in Mexico, which are attended by foreigners, apply the "0" rate benefit, which represents savings of 16% in food and beverages for banquet services and charges for room. This benefit will apply to foreign participants to Congresses, Conventions, Fairs and Exhibitions and is subject to changes without prior notice as it is a Federal Law.


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2 Bed, 2 Bath, Approx 1200 Sq Ft
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ZLI's Wild Planetarium Project helps K-12 students to achieve S.T.E.A.M. goals in scientific and cultural studies, while advancing diversity & inclusion mandates for a sustainable future. ZLI's Wild Planetarium Project offers an exciting S.T.E.A.M. program available nowhere else.
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REIMBURSEMENTS AND CORPORATE FUNDS. HC. "A dominant Texas engineering company led by a former University of Texas regent has agreed to a $1.6 million fine because top-level staffers facilitated hundreds of thousands of dollars of illegal campaign contributions." DOJ Release. … Continue reading
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AD QUESTIONS. WT. "Photographs and video from Sen. Gary Peters' campaign website have surfaced on a 'dark money' group's new pro-Peters ad, raising questions about illegal coordination between the groups." DEFAMATION LAWSUIT. CN. "Roy Moore, the Republican candidate who lost … Continue reading


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Toys Link Collectible Tin Red Face Robot
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Red Face Wind-up Tin Toy Robot
Wind him up, cool metal details, wire hair etc... Standing at 15 cm tall.
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Please note: This tin toys are pure nostalgia and collector articles and in today's sense no toys for children according to legal safety regulations for toys. As replicas of toys of the past, these articles cannot meet modern requirements without losing their authentic character.
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A One-Sided Phone Conversation
The name's Dawes, Jim Dawes. Now, the story I'm about to tell consists of very confidential material, so don't spill the beans. I work at the FBI as the person who listens in on the phone conversations (in case ordinary people are plotting to impact our country in a negative way). Now, I'm the newest recruit so I got stuck with the junk. By junk, I mean the listening machine where you can only hear one side of the phone conversation. Unfortunately, I had not been told of this problem from my co-worker, Bill Kwonkski. So, I carried on with my job. The first day that I used the machine was a day to remember––for me, Bill, and the entire FBI. I sat down on that Tuesday morning, thinking it would be another splendid day. I picked up my coffee and started chatting to Bill. Suddenly, out of the blue, the listening machine started to vibrate. That means that there is a suspicious call on the line. So I picked up the phone and started to listen:
Anonymous Caller: Hello Sweetie, how are you?
Anonymous Caller: That's wonderful. Are the dinner plans ready yet?
Anonymous Caller: Lovely, We should only be there for a bit.
Anonymous Caller: Oh no, everybody should be fine.
Anonymous Caller: This dinner will knock them cold.
Anonymous Caller: Oh, by the way, we're having Chinese.
Anonymous Caller: I don't think he'll mind if we don't say grace.
Anonymous Caller: Bo and Sunny won't miss him too much.
Anonymous Caller: Ok. See you on Monday
As you can see, it was a harmless phone call and the caller had no malicious intentions whatsoever. I sat back down at my desk and continued to talk with Bill. Two days later, the phone call came back to haunt me in the worst way possible. THE PRESIDENT, THE POPE, AND THE CHINESE FOREIGN MINISTER HAD BEEN KIDNAPPED!!! It didn't take too long to put two and two together, and I realized that I was the one responsible for all this. I had ignored the phone call. The dinner they had been talking about on the phone was the dinner that the president had hosted. I hoped that nobody else would notice what I had done. I decided to play it quiet. Unfortunately, Bill Kwonski remembered and he reported me to the head of the FBI. Being the FBI, they fired me and Bill in order to keep things secret.
Three years later, Bill married a nice lady by the name of Betty Gron. Betty and Bill decided to combine their last names and they had a cute baby boy by the name of Rob Gronkwonski, (who got really into football). As for me, I got really interested in 60's music and started a club in St. Louis. That day, I learned to never underestimate things. Because if you do, you might end up in a place or state that you never would have expected.
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Fi Shailes
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Dale Jr. went on the Dan Patrick Show where he said he wants to be in a "Mike Helton" role someday.
It's literally NASCAR fan's dream. Southerners have been saying this for years. God, Dale Jr, Country. Now they can add President to Dale's name. Albeit NASCAR President, he's still the President of something and that's all that matters to most people. They'll make flags, coffee cups, teddy bears, all to celebrate their new fearless leader.
Dale Jr. admitting that he wants to be President is actually a huge step to making it happen. Like a 12 step program the first step is admission. Dale has accomplished that first step. Being President of NASCAR would certainly be something.
Would Dale Jr. Make A Good President?
Talking about being President is really easy, being President is really hard. Look at that ole' boy in the White House. Any jabroni can sit on their couch and fire off some tweets. Not everyone can run an actual country. Granted NASCAR isn't a country but you have to deal with the most insane, illiterate, passionate fan base in the country.
Dale can do it though.
The people love Dale Jr. Everyone loves him, he's just a likable guy for the most part. His ideas are the general consensus of what people want. He's honest to a fault at times, no more hiding behind the NASCAR shield. Dale would come right out and say "we fucked up."
Also he would immediately make the competition better. Bye bye front splitters, the next season would be the return of valance. Dale would get the series back to the days of old when cars weren't so dependent on front downforce.
Honestly that's the biggest thing NASCAR fans want. Race cars that aren't so aero dependent that a small bump will ruin your day. A bump against the wall Denny Hamlin at Phoenix style will destroy your car. As President Dale Jr will look to make NASCAR strong again.
All rise for President Dale Jr.
Follow us on Twitter @ApexOff


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Cold and Flu articles
The 3-stage fightBy Nina Wagner on November 1, 2019
Are you eyeing up echinacea and elderberry now that cold and flu season is fully underway? Good. That's wise of… Continue reading...
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No one looks forward to being sick, but most of us expect to fall prey to a cold or flu… Continue reading...
Need a Boost?By Tracy Peternell on November 1, 2016
Our bodies are amazing at healing themselves through a positive lifestyle and good nutrition, but supporting the immune system is… Continue reading...
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Getting sick is the worst, especially when it happens again and again during fall and winter. If it… Continue reading...
5 Reasons to Stock up on Echinacea This SeasonBy alive Editorial on November 7, 2015
As cold and flu season begins ramping up, millions of people are again wondering how to best protect themselves from… Continue reading...
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We've already seen Paloma Faith rock some pretty wacky looks for London Fashion Week, and, now, the star is showing off her style in Italy, too! At the Max Mara show on September 19th, Paloma was working a retro beauty look on the front row.Fifties hair
Paloma's hair was styled into three large rolls, a decidedly fifties style! She kept the side of her hair smooth to give her quiff all the more impact. To get her look, use some retro hair rollers and add plenty of hairspray!Cat eye
Cat eyes are a beauty staple. Paloma's exaggerated flicks opened up her eyes and maximised the vintage appeal of her beauty look. Her mascara, pink lips and beauty spot finished the look beautifully. She looked like a redhead Marilyn!Pretty in pink
The retro theme didn't stop with Paloma's hair and make-up. She dressed herself in a deliciously fifties outfit comprised of a pink blouse, a pink pencil skirt, a pink coat and a very pink bag. We'd normally say that wearing all of the same colours is not OK but that seems to be Paloma's speciality and she totally pulled it off.
So there you have it, a good old old-fashioned makeup look and some great retro style!


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Psychic Killer Ray Danton
Published Dec 08, 2008Why rent the DVD release of 1975's Psychic Killer instead of another new release from last week - Hancock, perhaps? Certainly we've been overwhelmed by enough Hancock marketing that it seems like required viewing. Both movies are quite bad. But while Psychic Killer isn't as enjoyably bad as a Plan 9 From Outer Space or Sleepaway Camp 3: Teenage Wasteland, it's at least bad in a variety of interesting ways. Hancock is just bad in the modern-Blockbuster way that's really boring. Arnold Masters is wrongly incarcerated after a doctor is murdered. A confusing series of events endow him with psychic powers. He uses these to murder enemies from the comfort of his easy chair using astral projection. Yes, astral projection; it's not just for DMT-smoking teenage mystics and cough syrup chugging dissociative drug users anymore. The killing starts and we're treated to concise, self-contained murder segments worth watching for any horror buff. The real comic highlight is an incredibly serious seven-minute speech a professor gives on psychic ability. He ends his preachy message on the potential of the human mind with, "Have you ever had a stab of anguish for a dear loved one, and the very next day... the fateful telegram?" The music swells and the hard-boiled cop suddenly understands psychic phenomena on a profound, personal level. It has the look and feel of an ambitious Roger Corman production, with colourful performances and stylized camera work. Long zooms and unconventional lighting and editing remind us why bad movies from this period are so engaging: even the schlockiest genre pictures would often emulate the look of Cassavetes or Truffaut, resulting in rich aesthetics that while flawed are a visual treat in today's bland landscape of digital effects and glossy HD supermen. Despite a prolonged nude scene in which a nurse sexually harasses a paralyzed man, a dozen odd murders straight from any slasher flick and plenty of standard R-rated fare, Psychic Killer somehow got a PG rating from the violence-tolerating, lesbian-fearing MPAA. Critics often use Psychic Killer as an example of the MPAA's unjust ratings. (Dark Sky)


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Gateshead Council reject North East 'devo deal'
Gateshead Council's cabinet has rejected plans to bring devolved powers and an elected mayor to the North East.
All seven councils which form the North East Combined Authority are deciding whether to approve the deal.
Northumberland County Council approved the proposal, subject to certain conditions.
Sunderland, Newcastle, Durham, North Tyneside and South Tyneside will make their decisions later this week.


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Over Easter weekend, 17 National and 16 National traveled down to Philadelphia for the Northeast Qualifiers. They played a combined 33 sets of volleyball against teams from all over the country, and as far away as California.
Of special note were the officials who commented that Maine Juniors' work teams were amongst the best they'd worked with. We are so proud of our teams who work hard both on and off court to be their best. Thank you 17N & 16N for so well representing Maine on a national stage!


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user profile | dashboard | imagewall | contact user
About Me:Darren P Millar
I'm a DJ from Aberdeen (Scotland) that's about me for just now!
Twitter - href="http://www.gripmyshitter.com" target="_blank">Grip My Shitter - QIK - Flickr - href="http://www.darrenmillar.net" target="_blank">Blog - href="http://www.thisisdesignuk.com" target="_blank">My Work - href="http://www.darrenmillar.com" target="_blank">DJ - Bebo - Camera Man - href="http://www.denisemilani.com" target="_blank">Denise Milani - Last FM - Wishlist - My Mart - Lomo Home - €urotrip - Miss LaLa - Moblog Hotel - Mixwit - I Heart It - Brightkite - Regal Clothing
My Pet Hate: is TUESDAY, everything that has ever gone wrong in my life has happened on a TUESDAY! I hate it, it's now my day off so that i don't have to face the world! Don't make me have to suffer TUESDAY by asking me about it!!! I also dislike girls who drink pints, smoke and generally act like men!!
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My favorite!
Hi, I really like this picture!!! Mind if I use it for my DiDA project at school? I'll mention your blog, so the examiner knows it is yours. Thanks!
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1. Place each fish fillet on a piece of lightly oiled foil large enough to completely enclose each fillet.
2. Combine spring onions, ginger, garlic, soy, rind, juice and sugar in a small jug. Divide mixture among fish pieces and spread over each. Gather corners of the foil and pull over the fish, twist to enclose securely.
3. Preheat and oil a grill plate or barbecue on high. Cook parcels for 5 minutes or until fish is cooked as desired.
4. Meanwhile, boil, steam or microwave green vegetables until tender, and drain. Season to taste.
5. Serve salmon with vegetables on the side.


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This site is part of the Informa Tech Division of Informa PLC
This is operated by a business or businesses owned by Informa PLC and all copyright resides with them. Informa PLC's registered office is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 8860726.
Reconstruct, an innovative startup using drones, machine learning, and the cloud to manage construction projects better, chose Oracle for its cloud needs. This note explores why.


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The game was Mississippi State versus the University of Mississippi. The game? The annual Egg Bowl for the state rivalry.
The game was going back and forth or like many would categorize it as a "rollercoaster" when Mississippi State Rebels' WR Elijah Moore, scored a last-second score that looked like it would send the game to overtime with the extra point.
But Moore's end zone antics seemed to have cost his team field position for the extra point. As you will see in the video below, Moore raised his leg to simulate a dog urinating and the referee threw a flag which ended up costing his team the game when he drew a 15-yard unsportsmanlike penalty for a TD celebration.
Watch the video above, via PSC Highlights
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