BACKGROUND
natural antisense transcripts  are a class of endogenous coding or non-coding rnas that have sequence complementarity to other rnas in the cell.
cis-nats are transcribed from the opposite strands of the same genomic locus, in which case the sequence complementarity between two transcripts is directly related to the overlap in locations of the corresponding genes on the sense and antisense strands.
in contrast, trans-nats are transcribed from different genomic loci.
cis-nats usually have a long perfect complementary overlap between the sense and antisense transcripts, whereas the trans-nats often have short and imperfect complementarity.
recent genome-wide analyses revealed a surprisingly widespread existence of nats in eukaryotic genomes  <cit> , which suggests that nats may have evolved as a common regulatory mechanism for gene expression.
approximately 22–26% of human genes  <cit> ,  <dig> –29% of mouse genes  <cit> ,  <dig> – <dig> % of drosophila genes  <cit> ,  <dig> % of arabidopsis genes  <cit>  and 7% of rice genes are overlapping  <cit>  and form cis-nats.
recently, many new cis-nats were identified from the massively parallel signature sequencing  datasets of human and mouse nats  <cit> .
the authors suggested that alternative polyadenylation and retroposition may account for the origin of a significant number of functional sense-antisense pairs in mammalian genomes.
in spite of large intergenic spaces, many genes are still overlapping, such a genomic arrangement must be functionally beneficial.
nats are involved in diverse physiological processes, including pathophysiological processes in human diseases  <cit> .
despite the widespread of nats in eukaryotic genomes, the mechanisms of their regulation are still largely unknown.
nats have been implicated in various aspects of expression regulation of eukaryotic genes, including transcriptional interference  <cit> , rna masking-induced alternative splicing  <cit> , x chromosome inactivation  <cit> , genomic imprinting  <cit> , rna editing  <cit>  and small rna-induced gene silencing  <cit> .
recent analysis showed that 47% of the human imprinted genes in the igc database are arranged as nats  <cit> .
in small rna-induced gene silencing pathways, double-stranded rnas  can be processed by rnase iii enzymes known as dicers or dicer-like proteins into small interfering rnas   <cit> .
the sirnas are then incorporated into the argonaute-containing rna-induced silencing complex , to guide the cleavage of complementary mrnas, or argonaute-containing rna-induced transcriptional silencing complex , to guide chromatin modifications  <cit> .
co-expression of overlapping sense/antisense transcripts could potentially form dsrnas, which may be recognized by dicer or dicer-like proteins and processed into sirnas.
recently, we found two pairs of cis-nats in arabidopsis that generate sirnas from the sense strand , which then target the antisense strand for cleavage  <cit> .
in these two cases, the sense transcript is induced by abiotic or biotic stress and the resulting nat-sirnas cause the silencing of the constitutively expressed antisense transcript.
these results suggest that the inverse changes in gene expression within a nat pair , may be a diagnostic feature of rnai regulation of nats.
a genome-wide analysis of human cis-nats has indicated that the genes within a nat pair tend to be inversely expressed and/or co-expressed more frequently than expected by chance  <cit> , although whether any nats are regulated by rnai in humans is unclear.
the proportion of inversely expressed cis-nats in the arabidopsis genome is unknown, and the extent of small rna-mediated gene regulation within the nat genes is completely unknown in any organism.
recent studies employing high throughput sequencing of small rnas have identified endogenous small rnas matching many protein coding genes in arabidopsis  <cit> .
we were interested in determining whether cis-nat genes are equally prone to small rna regulation, or are deficient or enriched for small rna matches.
the identification of nats with inverse expression patterns and small rnas that match the nats would provide an indication of rnai regulation of the nats.
to determine the extent of regulation of cis-nats by rnai, we conducted a genome-wide analysis of arabidopsis cis-nat gene expression and identified a large number of sirnas that match these cis-nats.
we found a subgroup of cis-nats with negative co-regulation between their sense and antisense partners that are enriched for small rna matches.
in general, cis-nat protein-coding genes have a slightly higher frequency of small rna matches than non-nat protein-coding genes.
we conclude that many cis-nat genes may be regulated by sirnas in response to various environmental or developmental cues.
RESULTS
identification of arabidopsis cis-nats with inverse expression changes in response to various environmental and developmental conditions
co-expression of sense and antisense transcripts within a nat pair could potentially form dsrnas, which could be processed into small rnas and cause silencing of the antisense transcript.
we hypothesized that the expression of many nat genes may be induced under specific environmental or developmental conditions to avoid the cost of constant silencing caused by sense-antisense transcript pairing.
to test this hypothesis, we identified all the cis-nats in the arabidopsis genome and conducted computational analysis of large-scale microarray expression data from public databases.
we searched the arabidopsis information resource  and identified  <dig> nat pairs with a minimum overlap of  <dig> base pairs , including both cis- and trans-nats.
when loci of several splice variants overlapped, gene models with the longest overlapping region were chosen.
of the  <dig> pairs,  <dig> pairs are cis-nats, that is, the two genes within the nat pair are located on the opposite strands of the same genomic locus.
these cis-nats include  <dig> pairs with overlap between protein-coding genes,  <dig> with overlap between protein-coding and t/sn/snorna genes,  <dig> involving trans-acting sirna  primary transcripts and  <dig> pairs involving transposon genes .
most of the cis-nats  are arranged in convergent orientation, with the 3' ends overlapping.
the overlap between sense and antisense transcripts in  <dig> pairs involves introns.
interestingly, we found  <dig> genes overlapping with two distinct genes on the opposite strands .
in this study, we mainly focused on the protein-coding cis-nats.
pc: protein coding gene
to determine the extent of small rna-mediated gene silencing in regulation of cis-nat genes, we performed correlation and differential expression analyses of  <dig> affymetrix ath <dig> genome arrays derived from  <dig> different experiment series from the atgenexpress  <cit>  and geo sites  <cit>  .
we performed statistical analysis of differentially expressed genes only on the experiment sets with two or more replicates.
the selected replicated experiment sets included mutant and tissue comparisons, as well as sample sets that were treated with different abiotic stresses, biotic stresses, chemicals and hormones .
all nat pairs represented on the ath <dig> chip by ambiguous probe sets  were excluded from the subsequent steps of the analysis.
the remaining  <dig> nat pairs with unambiguous probe sets were used for correlation and differentially expressed gene  analyses.
to identify global negative co-regulation effects between nat pair members, we calculated the pearson and spearman correlation coefficients for the expression profiles of sense and antisense partners of each gene pair.
we used the averaged intensity values between replicates of the total dataset  for this analysis.
the obtained correlation coefficients provide a measure of the similarity of expression profiles.
correlation values close to + <dig> indicate positive correlation, values close to  <dig> imply no correlation and values close to - <dig> indicate negative correlation.
we identified  <dig> nat pairs with a pearson correlation coefficient ≤ - <dig>  and  <dig> pairs with a spearman correlation coefficient ≤ - <dig> ; with a correlation cutoff of ≤ - <dig> , we identified  <dig> and  <dig> pairs, respectively.
interestingly, about 85% of the identified negatively co-regulated gene pairs were also identified by the differential expression analysis  described later.
when the averaged gene expression levels across all experiments are divided into the three expression classes high, medium and low, 52% of the negatively correlated genes from the pearson method are expressed at high, 35% at intermediate and 13% at low levels .
for the  <dig> pairs from the spearman method it is 47%, 38% and 15%, respectively.
these results indicate that the many negatively correlated nat pair members are expressed at intermediate or high levels.
the mean pearson and spearman correlation coefficients with their standard deviations for randomized , nat and all non-overlapping gene pairs .
a two-sample t-test was used to calculate p-values for differences in distribution of coefficients between the cis-nat dataset and the other two datasets.
we also attempted to identify specific inverse expression changes between nat sense and antisense transcripts by deg analysis using as cutoff an adjusted p-value of the limma package of ≤  <dig>  and a fold change of at least  <dig>  <cit> .
this stringent strategy identified  <dig> pairs with at least one inverse expression change at specific comparisons .
of these  <dig> pairs,  <dig> had a negative  and  <dig> a positive  pearson correlation coefficient.
because the silencing of an antisense target may occur at later time points or stages, we also identified slow expression responses with inverse fold changes between sense and antisense transcripts anywhere  within a given experiment set using the same cutoff parameters .
this less stringent approach identified  <dig> pairs with at least one inverse expression change anywhere within an experiment set .
of these  <dig> pairs,  <dig> had a negative  and  <dig> a positive  pearson correlation coefficient.
thus, many nat pairs display inverse expression changes in response to specific environmental or developmental cues.
however, as shown in table  <dig>  the number of observed nat gene pairs with opposing expression changes is not significantly greater than that obtained from randomized datasets consisting of artificially joined gene pairs.
the result indicates that the deg analysis as applied here cannot be used to reliably identify inversely regulated cis-nats.
nevertheless, the nat pairs identified by the deg analysis as having inverse expression profiles may still be useful for experimental validation as putatively inversely regulated nats.
the number  of cis-nat pairs with opposite differential expression changes are given for the stringent  and the less stringent  approach.
the same counts were calculated for randomized gene pairs .
their mean values and standard deviations  are provided.
the p-values represent the number of times the randomized data generated a value ≥ the observed value  divided by  <dig>
in summary, the data from both correlation analysis and differential expression study show small differences in opposite expression patterns between cis-nat, randomized and non-overlapping gene pair samples.
consistent with results of a recent study of expression patterns of nats  <cit> , these results indicate that negative regulation mechanisms may not be shared by most cis-nat pairs but are restricted to a subgroup of this gene class that exhibits opposite expression profiles.
alternatively, many cis-nats might display subtle negative co-regulations only under specific developmental stages or environmental conditions.
in the latter case, the associated responses can only be detected by detailed differential gene expression analyses and not by global correlation analyses between pair members.
in addition, there may be nat pairs in which both of the two overlapping genes show an overall positive correlation, but one of the gene members may be strongly induced only under specific conditions.
the specific induction of the one member could reach a threshold and result in the down-regulation of its antisense partner by the formation of nat-sirnas.
finally, we would like to point out that small rna-mediated gene regulation of nats occurs only when both transcripts are expressed within the same cell.
many nats can generate sirnas
our recent studies suggest that the expression of nats may be regulated by endogenous sirnas  <cit> .
we searched several publicly available datasets – the arabidopsis small rna project   <cit>  , mpss  <cit>  and a dataset recently published by bartel and coworkers   <cit>  – for small rnas with 100% sequence match to arabidopsis cis-nats.
nats that match transposon, retrotransposon, rrna, trna, snrna or snorna genes were removed prior to this analysis.
we identified  <dig> unique small rnas, including  <dig> and  <dig> small rna sequences from the asrp and db datasets, respectively, and  <dig> unique signatures from the mpss dataset , which match a total of  <dig> nat genes .
among them,  <dig> small rnas match  <dig> nat overlapping regions .
agi genes: from tair  <dig> .
pc genes: protein-coding genes.
although current asrp, mpss and db small rna datasets were generated from different developmental stages/tissues  or from different small rna biogenesis mutants, very little stress-treated material was included  <cit> .
because many nats appear to be under the regulation of various abiotic and biotic conditions , we expected to identify more nat associated sirnas from plants treated with various stress conditions.
we performed a pilot small rna profiling experiment on stress-treated plants using  <dig> sequencing  <cit> .
the stress conditions included both abiotic  and biotic stresses  strain dc <dig> and dc <dig> carrying an avirulence gene avrrpt <dig>  or fungi alternaria brassicicola and botrytis cinerea).
of  <dig>  unique small rnas with a blast hit against the arabidopsis genome , we identified  <dig> new small rnas that match the nats but are not present in the asrp, mpss or db datasets.
the new small rnas from the stress libraries match  <dig> nat genes, with  <dig> located in  <dig> distinct overlapping regions .
these stress library-specific small rnas match  <dig> new nat genes .
these results, together with the little overlap among these datasets, suggest that the small rna population identified from these datasets is far from saturation.
abiotic stress-treated libraries
abio1: cold treatment, 4°c
abio2: draught-treatment and aba treatment
abio3: salt treatment and copper treatment
abio4: uv treatment and heat treatment
bio1: pseudomonas syringae dc <dig>  and dc <dig>  infection
bio2: a.b., and b. c. infection
in total, we identified  <dig> distinct small rnas from asrp, mpss, db and the stress-treated dataset, which together match  <dig> of  <dig> protein-coding cis-nat genes .
this value is slightly higher than the proportion of non-nat protein-coding genes with small rna matches .
the difference in hit frequency is significant .
the  <dig> cis-nat genes with small rna hits comprise  <dig> cis-nat pairs  in arabidopsis .
this number is comparable with the probability that for two randomly paired non-nat protein-coding genes, at least one will have a matching small rna .
the analysis of the subset of nats with negatively correlated expression patterns  showed increased frequency of small rna hits .
compared to nat pairs with pearson correlation score ≥ - <dig> , the former has a higher frequency of small rna hits both for the overlapping region  and for full-length nats .
compared to the small rna hit frequency for random pairs formed from non-nat protein-coding genes, the full-length nat hit frequency of the subgroup is higher  but not statistically significant .
the p-value for the enrichment in the number of overlap regions  was  <dig> , and for the enrichment in the number of hits in the full-length nat region  was  <dig> .
some putative nat-sirnas may regulate more than one antisense gene
in some cases, the sirnas generated from cis-nats match not only the genes in cis but also other mrnas transcribed from non-overlapping loci in trans.
figure  <dig> shows an example of a small rna with complementarity to three different transcripts, which thus may potentially target all of them.
these three transcripts are highly similar to each other and all encode udp-glycosyl transferases.
an sirna signature identified from the mpss small rna database matched the overlapping region of a cbs domain-containing protein gene  and one of the udp-glucosyl transferase genes  and has the potential to silence all three of the udp-glycosyl transferase genes, since all three of the transcripts have a 100% complementary match to the sirna.
interestingly, two ta-sirna primary transcripts also overlap with other genes.
tas1c overlaps with a radical sam domain-containing protein gene and generates many sirnas  from both the sense and antisense strands of the locus, with some hitting the overlapping region.
tas <dig> also overlaps with a gene with protein-coding potential.
many sirnas , including several ta-sirnas that regulate pentatricopeptide-repeats protein genes in trans, are generated from both the sense and antisense strands of this locus.
among the putative nat-sirnas identified,  <dig> have complementarity with and may target more than one gene.
we identified  <dig> putative nat-sirnas that hit two or more genes within a gene family .
consistent with our results, a recent study of arabidopsis trans-nats identified more than  <dig> nat genes with both cis and trans partners  <cit> .
therefore, cis-nat-generated sirnas may play a broader role in gene regulation beyond that of the overlapping genes.
they may regulate other gene family members or a group of genes with conserved small rna target sites in trans and thus may be involved in complex gene regulatory networks.
functional annotation of arabidopsis cis-nats
we used enrichment analysis of gene ontology  terms to functionally analyze the identified cis-nats  <cit> .
the entire cis-nat set and the  <dig> pairs with inverse expression changes underwent statistical testing based on the hypergeometric distribution.
we aimed to identify overrepresented terms by assigning a p-value to every node in the go network  <cit> .
in both sample sets the most highly enriched go terms, among a total of  <dig> go slim categories, were "catalytic activity" in the molecular function ontology with  <dig> and  <dig> sample matches at a node with  <dig> associated genes , and for the cellular component ontology it was "plastid"  and "mitochondria" .
figure  <dig> shows the relative distribution of cellular-component go categories among cis-nat genes, non-nat genes and all genes in the arabidopsis genome.
interestingly,  <dig> nat genes  are associated with plastids and  <dig> with mitochondria .
these results indicate that 33% of the nat genes are involved in processes associated with organelles having presumed prokaryotic origins.
computational predictions of subcellular targeting by the targetp program  <cit>  generated similar results .
to identify gene pairs in which the sense and antisense partners have related functions and locations, we searched all nodes in the three ontologies for common terms between the two members of each nat pair.
when considering only relatively specific go terms with  <dig> or more ancestor nodes from the root of each ontology, we found  <dig> nat pairs sharing at least one common go term.
randomly selected pairs show a significantly lower number of common go terms .
the detailed go analysis of gene pairs is available in additional file  <dig>
discussion
eukaryotic genomes feature large intergenic spaces, but contain a significant number of overlapping genes that encode cis-nats.
the specific overlapping arrangement is presumed to have physiological benefits.
however, the general mechanisms of cis-nat regulation are still unknown.
our recent studies provided two examples of nat-sirnas with an important role in regulating cis-nat expression in response to abiotic and biotic stress  <cit> .
here, we performed a genome-wide analysis of arabidopsis cis-nats and discovered a group of nat pairs with an inverse expression pattern between the sense and antisense transcripts in response to various stress treatments and/or at different developmental stages.
this negative-expression correlation within a nat pair, together with the finding that more than  <dig> nat pairs share a common go term, suggests that the overlapping arrangement is important for expression regulation of at least a subgroup of nat genes in certain cellular processes or in response to stresses.
we hypothesized that the induced expression of one transcript may pair with the existing antisense transcript within the same cell to form dsrna, which can be processed into nat-sirna.
these nat-sirnas can cause silencing of the antisense transcript.
although the negative-expression correlation within a nat pair could also be caused by transcriptional interference, the identification of about  <dig> putative nat-sirnas from our stress-treated small rna dataset and current mpss, asrp and db small rna databases, together with the observation that small rnas are enriched in the overlapping regions of negatively correlated nat pairs compared with non-negatively correlated pairs, strongly suggests that sirna-mediated gene silencing may be one of the major molecular mechanisms of gene expression regulation of at least a subgroup of cis-nats.
current small rna cloning strategies may have not been able to identify nat-sirnas present at very low levels or induced only transiently, by treatments of short durations or by a narrow window of treatment levels.
this suggestion is also supported by little overlap of identified nat-sirna candidates from different datasets.
we expect to identify more nat-sirnas by deeper coverage sequencing of small rnas from plants treated with more stress conditions and more defined time intervals.
dsrnas are formed during plant virus replication and transgene expression by viral rna-dependent rna polymerase  or plant endogenous rdrs  <cit> .
these dsrnas are processed by dicer-like proteins and generate sirnas from both strands and cover the whole double-stranded region to induce silencing.
although the pairing of sense and antisense nat transcripts also forms dsrnas and triggers sirna formation, our published work on three pairs of nats suggests that nat-sirnas are often generated from one specific strand and/or from one specific site to induce silencing of only one strand of the nat pair  <cit> .
to determine whether this is a general observation, we examined all the overlapping regions that have putative nat-sirna hits, and revealed that 83% of these regions are targeted by strand-specific small rnas.
about 17% of these regions have small rna hits on both strands, which can potentially silencing both the sense and antisense transcripts simultaneously.
these results indicate that majority of the nat-sirnas regulate only one transcript within a nat pair and suggest that the formation of nat-sirnas may require additional sequence and/or structural features of the overlapping transcripts or additional factors.
this suggestion is supported by our recent study of a bacteria inducible nat-sirna, nat-sirnaatgb <dig>  whose induction by the bacterial pathogen p.s.t.
carrying the avirulence gene avrrpt <dig> required not only the induction of the sense gene atgb <dig> but also the presence of cognate resistance gene rps <dig> and its resistance signaling component ndr <dig>  <cit> .
the biogenesis of this nat-sirna is also under the control of dicer-like  <dig>  rdr <dig>  rna polymerase iva, hyl <dig> and hen <dig>  <cit> .
this finding indicates that multiple layers of control exist in nat-sirna biogenesis and that the induction of the sense transcript of a nat pair is necessary but may not be sufficient to induce some of the nat-sirnas.
our recent studies of stress-induced nat-sirnas demonstrated that the biogenesis of these nat-sirnas also depends on rdr <dig> and poliva subunit nrpd1a, the components required for rna replication  <cit> .
thus, dsrna formed by sense-antisense pairing might involve a secondary rna replication loop to form nat-sirnas.
this rna amplification step may extend beyond the overlapping region to form sirnas outside the overlapping region.
we hypothesize that these nat-sirnas may ensure that the antisense gene is silenced.
furthermore, "tiling" microarray studies to assay transcript levels throughout the genomes have revealed that more than 30% of the arabidopsis genome and about 60–80% of mammalian genomes can produce transcripts from both strands  <cit> .
about  <dig> genes in arabidopsis showed significant antisense rna expression  <cit> .
interestingly, a recent study of rice involving robust-long serial analysis of gene expression revealed that many antisense transcripts were induced after the infection of a fungal pathogen magnaporthe grisea, which induces rice blast disease  <cit> .
therefore, antisense transcription is not only limited to the nats but also applies to many non-nat genes.
antisense transcription potentially forms dsrnas, which can be recognized by rnai machinery and generate small rnas to guide gene expression regulation.
that antisense transcription can be induced by pathogen infection is of particular interest.
we have discovered that many small rnas are also induced by pathogen infection .
we are currently exploring whether some of these pathogen-induced small rnas may be generated by the induction of antisense transcription outside the nat regions.
CONCLUSIONS
we have performed a comprehensive gene expression analysis of arabidopsis cis-nats and discovered that a significant fraction of the sense and antisense partners of cis-nats are anti-correlated, with inverse changes in expression in response to environmental or developmental cues.
we identified nearly  <dig> unique sirnas that match cis-nat genes.
the sirnas are enriched in the overlapping regions of negatively correlated nat pairs, which supports our hypothesis that sirna-mediated gene silencing may be one of the important mechanisms in gene regulation of some nat genes.

 natural antisense transcripts are a class of endogenous coding or non-coding rnas that have sequence complementarity to other rnas in the cell. cis-nats are transcribed from the opposite strands of the same genomic locus, in which case the sequence complementarity between two transcripts is directly related to the overlap in locations of the corresponding genes on the sense and antisense strands. in contrast, trans-nats are transcribed from different genomic loci. cis-nats usually have a long perfect complementary overlap between the sense and antisense transcripts, whereas the trans-nats often have short and imperfect complementarity. recent genome-wide analyses revealed a surprisingly widespread existence of nats in eukaryotic genomes, which suggests that nats may have evolved as a common regulatory mechanism for gene expression. approximately 22–26% of human genes, –29% of mouse genes, – % of drosophila genes, % of arabidopsis genes and 7% of rice genes are overlapping and form cis-nats. recently, many new cis-nats were identified from the massively parallel signature sequencing datasets of human and mouse nats. the authors suggested that alternative polyadenylation and retroposition may account for the origin of a significant number of functional sense-antisense pairs in mammalian genomes. in spite of large intergenic spaces, many genes are still overlapping, such a genomic arrangement must be functionally beneficial. nats are involved in diverse physiological processes, including pathophysiological processes in human diseases. despite the widespread of nats in eukaryotic genomes, the mechanisms of their regulation are still largely unknown. nats have been implicated in various aspects of expression regulation of eukaryotic genes, including transcriptional interference, rna masking-induced alternative splicing, x chromosome inactivation, genomic imprinting, rna editing and small rna-induced gene silencing. recent analysis showed that 47% of the human imprinted genes in the igc database are arranged as nats. in small rna-induced gene silencing pathways, double-stranded rnas can be processed by rnase iii enzymes known as dicers or dicer-like proteins into small interfering rnas. the sirnas are then incorporated into the argonaute-containing rna-induced silencing complex, to guide the cleavage of complementary mrnas, or argonaute-containing rna-induced transcriptional silencing complex, to guide chromatin modifications. co-expression of overlapping sense/antisense transcripts could potentially form dsrnas, which may be recognized by dicer or dicer-like proteins and processed into sirnas. recently, we found two pairs of cis-nats in arabidopsis that generate sirnas from the sense strand, which then target the antisense strand for cleavage. in these two cases, the sense transcript is induced by abiotic or biotic stress and the resulting nat-sirnas cause the silencing of the constitutively expressed antisense transcript. these results suggest that the inverse changes in gene expression within a nat pair, may be a diagnostic feature of rnai regulation of nats. a genome-wide analysis of human cis-nats has indicated that the genes within a nat pair tend to be inversely expressed and/or co-expressed more frequently than expected by chance, although whether any nats are regulated by rnai in humans is unclear. the proportion of inversely expressed cis-nats in the arabidopsis genome is unknown, and the extent of small rna-mediated gene regulation within the nat genes is completely unknown in any organism. recent studies employing high throughput sequencing of small rnas have identified endogenous small rnas matching many protein coding genes in arabidopsis. we were interested in determining whether cis-nat genes are equally prone to small rna regulation, or are deficient or enriched for small rna matches. the identification of nats with inverse expression patterns and small rnas that match the nats would provide an indication of rnai regulation of the nats. to determine the extent of regulation of cis-nats by rnai, we conducted a genome-wide analysis of arabidopsis cis-nat gene expression and identified a large number of sirnas that match these cis-nats. we found a subgroup of cis-nats with negative co-regulation between their sense and antisense partners that are enriched for small rna matches. in general, cis-nat protein-coding genes have a slightly higher frequency of small rna matches than non-nat protein-coding genes. we conclude that many cis-nat genes may be regulated by sirnas in response to various environmental or developmental cues.  identification of arabidopsis cis-nats with inverse expression changes in response to various environmental and developmental conditions co-expression of sense and antisense transcripts within a nat pair could potentially form dsrnas, which could be processed into small rnas and cause silencing of the antisense transcript. we hypothesized that the expression of many nat genes may be induced under specific environmental or developmental conditions to avoid the cost of constant silencing caused by sense-antisense transcript pairing. to test this hypothesis, we identified all the cis-nats in the arabidopsis genome and conducted computational analysis of large-scale microarray expression data from public databases. we searched the arabidopsis information resource and identified nat pairs with a minimum overlap of base pairs, including both cis- and trans-nats. when loci of several splice variants overlapped, gene models with the longest overlapping region were chosen. of the pairs, pairs are cis-nats, that is, the two genes within the nat pair are located on the opposite strands of the same genomic locus. these cis-nats include pairs with overlap between protein-coding genes, with overlap between protein-coding and t/sn/snorna genes, involving trans-acting sirna primary transcripts and pairs involving transposon genes. most of the cis-nats are arranged in convergent orientation, with the 3' ends overlapping. the overlap between sense and antisense transcripts in pairs involves introns. interestingly, we found genes overlapping with two distinct genes on the opposite strands. in this study, we mainly focused on the protein-coding cis-nats. pc: protein coding gene to determine the extent of small rna-mediated gene silencing in regulation of cis-nat genes, we performed correlation and differential expression analyses of affymetrix ath genome arrays derived from different experiment series from the atgenexpress and geo sites. we performed statistical analysis of differentially expressed genes only on the experiment sets with two or more replicates. the selected replicated experiment sets included mutant and tissue comparisons, as well as sample sets that were treated with different abiotic stresses, biotic stresses, chemicals and hormones. all nat pairs represented on the ath chip by ambiguous probe sets were excluded from the subsequent steps of the analysis. the remaining nat pairs with unambiguous probe sets were used for correlation and differentially expressed gene analyses. to identify global negative co-regulation effects between nat pair members, we calculated the pearson and spearman correlation coefficients for the expression profiles of sense and antisense partners of each gene pair. we used the averaged intensity values between replicates of the total dataset for this analysis. the obtained correlation coefficients provide a measure of the similarity of expression profiles. correlation values close to + indicate positive correlation, values close to imply no correlation and values close to - indicate negative correlation. we identified nat pairs with a pearson correlation coefficient ≤ - and pairs with a spearman correlation coefficient ≤ -; with a correlation cutoff of ≤ -, we identified and pairs, respectively. interestingly, about 85% of the identified negatively co-regulated gene pairs were also identified by the differential expression analysis described later. when the averaged gene expression levels across all experiments are divided into the three expression classes high, medium and low, 52% of the negatively correlated genes from the pearson method are expressed at high, 35% at intermediate and 13% at low levels. for the pairs from the spearman method it is 47%, 38% and 15%, respectively. these results indicate that the many negatively correlated nat pair members are expressed at intermediate or high levels. the mean pearson and spearman correlation coefficients with their standard deviations for randomized, nat and all non-overlapping gene pairs. a two-sample t-test was used to calculate p-values for differences in distribution of coefficients between the cis-nat dataset and the other two datasets. we also attempted to identify specific inverse expression changes between nat sense and antisense transcripts by deg analysis using as cutoff an adjusted p-value of the limma package of ≤ and a fold change of at least. this stringent strategy identified pairs with at least one inverse expression change at specific comparisons. of these pairs, had a negative and a positive pearson correlation coefficient. because the silencing of an antisense target may occur at later time points or stages, we also identified slow expression responses with inverse fold changes between sense and antisense transcripts anywhere within a given experiment set using the same cutoff parameters. this less stringent approach identified pairs with at least one inverse expression change anywhere within an experiment set. of these pairs, had a negative and a positive pearson correlation coefficient. thus, many nat pairs display inverse expression changes in response to specific environmental or developmental cues. however, as shown in table the number of observed nat gene pairs with opposing expression changes is not significantly greater than that obtained from randomized datasets consisting of artificially joined gene pairs. the result indicates that the deg analysis as applied here cannot be used to reliably identify inversely regulated cis-nats. nevertheless, the nat pairs identified by the deg analysis as having inverse expression profiles may still be useful for experimental validation as putatively inversely regulated nats. the number of cis-nat pairs with opposite differential expression changes are given for the stringent and the less stringent approach. the same counts were calculated for randomized gene pairs. their mean values and standard deviations are provided. the p-values represent the number of times the randomized data generated a value ≥ the observed value divided by in summary, the data from both correlation analysis and differential expression study show small differences in opposite expression patterns between cis-nat, randomized and non-overlapping gene pair samples. consistent with results of a recent study of expression patterns of nats, these results indicate that negative regulation mechanisms may not be shared by most cis-nat pairs but are restricted to a subgroup of this gene class that exhibits opposite expression profiles. alternatively, many cis-nats might display subtle negative co-regulations only under specific developmental stages or environmental conditions. in the latter case, the associated responses can only be detected by detailed differential gene expression analyses and not by global correlation analyses between pair members. in addition, there may be nat pairs in which both of the two overlapping genes show an overall positive correlation, but one of the gene members may be strongly induced only under specific conditions. the specific induction of the one member could reach a threshold and result in the down-regulation of its antisense partner by the formation of nat-sirnas. finally, we would like to point out that small rna-mediated gene regulation of nats occurs only when both transcripts are expressed within the same cell. many nats can generate sirnas our recent studies suggest that the expression of nats may be regulated by endogenous sirnas. we searched several publicly available datasets – the arabidopsis small rna project, mpss and a dataset recently published by bartel and coworkers – for small rnas with 100% sequence match to arabidopsis cis-nats. nats that match transposon, retrotransposon, rrna, trna, snrna or snorna genes were removed prior to this analysis. we identified unique small rnas, including and small rna sequences from the asrp and db datasets, respectively, and unique signatures from the mpss dataset, which match a total of nat genes. among them, small rnas match nat overlapping regions. agi genes: from tair. pc genes: protein-coding genes. although current asrp, mpss and db small rna datasets were generated from different developmental stages/tissues or from different small rna biogenesis mutants, very little stress-treated material was included. because many nats appear to be under the regulation of various abiotic and biotic conditions, we expected to identify more nat associated sirnas from plants treated with various stress conditions. we performed a pilot small rna profiling experiment on stress-treated plants using sequencing. the stress conditions included both abiotic and biotic stresses strain dc and dc carrying an avirulence gene avrrpt or fungi alternaria brassicicola and botrytis cinerea). of unique small rnas with a blast hit against the arabidopsis genome, we identified new small rnas that match the nats but are not present in the asrp, mpss or db datasets. the new small rnas from the stress libraries match nat genes, with located in distinct overlapping regions. these stress library-specific small rnas match new nat genes. these results, together with the little overlap among these datasets, suggest that the small rna population identified from these datasets is far from saturation. abiotic stress-treated libraries abio1: cold treatment, 4°c abio2: draught-treatment and aba treatment abio3: salt treatment and copper treatment abio4: uv treatment and heat treatment bio1: pseudomonas syringae dc and dc infection bio2: a.b., and b. c. infection in total, we identified distinct small rnas from asrp, mpss, db and the stress-treated dataset, which together match of protein-coding cis-nat genes. this value is slightly higher than the proportion of non-nat protein-coding genes with small rna matches. the difference in hit frequency is significant. the cis-nat genes with small rna hits comprise cis-nat pairs in arabidopsis. this number is comparable with the probability that for two randomly paired non-nat protein-coding genes, at least one will have a matching small rna. the analysis of the subset of nats with negatively correlated expression patterns showed increased frequency of small rna hits. compared to nat pairs with pearson correlation score ≥ -, the former has a higher frequency of small rna hits both for the overlapping region and for full-length nats. compared to the small rna hit frequency for random pairs formed from non-nat protein-coding genes, the full-length nat hit frequency of the subgroup is higher but not statistically significant. the p-value for the enrichment in the number of overlap regions was, and for the enrichment in the number of hits in the full-length nat region was. some putative nat-sirnas may regulate more than one antisense gene in some cases, the sirnas generated from cis-nats match not only the genes in cis but also other mrnas transcribed from non-overlapping loci in trans. figure shows an example of a small rna with complementarity to three different transcripts, which thus may potentially target all of them. these three transcripts are highly similar to each other and all encode udp-glycosyl transferases. an sirna signature identified from the mpss small rna database matched the overlapping region of a cbs domain-containing protein gene and one of the udp-glucosyl transferase genes and has the potential to silence all three of the udp-glycosyl transferase genes, since all three of the transcripts have a 100% complementary match to the sirna. interestingly, two ta-sirna primary transcripts also overlap with other genes. tas1c overlaps with a radical sam domain-containing protein gene and generates many sirnas from both the sense and antisense strands of the locus, with some hitting the overlapping region. tas also overlaps with a gene with protein-coding potential. many sirnas, including several ta-sirnas that regulate pentatricopeptide-repeats protein genes in trans, are generated from both the sense and antisense strands of this locus. among the putative nat-sirnas identified, have complementarity with and may target more than one gene. we identified putative nat-sirnas that hit two or more genes within a gene family. consistent with our results, a recent study of arabidopsis trans-nats identified more than nat genes with both cis and trans partners. therefore, cis-nat-generated sirnas may play a broader role in gene regulation beyond that of the overlapping genes. they may regulate other gene family members or a group of genes with conserved small rna target sites in trans and thus may be involved in complex gene regulatory networks. functional annotation of arabidopsis cis-nats we used enrichment analysis of gene ontology terms to functionally analyze the identified cis-nats. the entire cis-nat set and the pairs with inverse expression changes underwent statistical testing based on the hypergeometric distribution. we aimed to identify overrepresented terms by assigning a p-value to every node in the go network. in both sample sets the most highly enriched go terms, among a total of go slim categories, were "catalytic activity" in the molecular function ontology with and sample matches at a node with associated genes, and for the cellular component ontology it was "plastid" and "mitochondria". figure shows the relative distribution of cellular-component go categories among cis-nat genes, non-nat genes and all genes in the arabidopsis genome. interestingly, nat genes are associated with plastids and with mitochondria. these results indicate that 33% of the nat genes are involved in processes associated with organelles having presumed prokaryotic origins. computational predictions of subcellular targeting by the targetp program generated similar results. to identify gene pairs in which the sense and antisense partners have related functions and locations, we searched all nodes in the three ontologies for common terms between the two members of each nat pair. when considering only relatively specific go terms with or more ancestor nodes from the root of each ontology, we found nat pairs sharing at least one common go term. randomly selected pairs show a significantly lower number of common go terms. the detailed go analysis of gene pairs is available in additional file discussion eukaryotic genomes feature large intergenic spaces, but contain a significant number of overlapping genes that encode cis-nats. the specific overlapping arrangement is presumed to have physiological benefits. however, the general mechanisms of cis-nat regulation are still unknown. our recent studies provided two examples of nat-sirnas with an important role in regulating cis-nat expression in response to abiotic and biotic stress. here, we performed a genome-wide analysis of arabidopsis cis-nats and discovered a group of nat pairs with an inverse expression pattern between the sense and antisense transcripts in response to various stress treatments and/or at different developmental stages. this negative-expression correlation within a nat pair, together with the finding that more than nat pairs share a common go term, suggests that the overlapping arrangement is important for expression regulation of at least a subgroup of nat genes in certain cellular processes or in response to stresses. we hypothesized that the induced expression of one transcript may pair with the existing antisense transcript within the same cell to form dsrna, which can be processed into nat-sirna. these nat-sirnas can cause silencing of the antisense transcript. although the negative-expression correlation within a nat pair could also be caused by transcriptional interference, the identification of about putative nat-sirnas from our stress-treated small rna dataset and current mpss, asrp and db small rna databases, together with the observation that small rnas are enriched in the overlapping regions of negatively correlated nat pairs compared with non-negatively correlated pairs, strongly suggests that sirna-mediated gene silencing may be one of the major molecular mechanisms of gene expression regulation of at least a subgroup of cis-nats. current small rna cloning strategies may have not been able to identify nat-sirnas present at very low levels or induced only transiently, by treatments of short durations or by a narrow window of treatment levels. this suggestion is also supported by little overlap of identified nat-sirna candidates from different datasets. we expect to identify more nat-sirnas by deeper coverage sequencing of small rnas from plants treated with more stress conditions and more defined time intervals. dsrnas are formed during plant virus replication and transgene expression by viral rna-dependent rna polymerase or plant endogenous rdrs. these dsrnas are processed by dicer-like proteins and generate sirnas from both strands and cover the whole double-stranded region to induce silencing. although the pairing of sense and antisense nat transcripts also forms dsrnas and triggers sirna formation, our published work on three pairs of nats suggests that nat-sirnas are often generated from one specific strand and/or from one specific site to induce silencing of only one strand of the nat pair. to determine whether this is a general observation, we examined all the overlapping regions that have putative nat-sirna hits, and revealed that 83% of these regions are targeted by strand-specific small rnas. about 17% of these regions have small rna hits on both strands, which can potentially silencing both the sense and antisense transcripts simultaneously. these results indicate that majority of the nat-sirnas regulate only one transcript within a nat pair and suggest that the formation of nat-sirnas may require additional sequence and/or structural features of the overlapping transcripts or additional factors. this suggestion is supported by our recent study of a bacteria inducible nat-sirna, nat-sirnaatgb whose induction by the bacterial pathogen p.s.t. carrying the avirulence gene avrrpt required not only the induction of the sense gene atgb but also the presence of cognate resistance gene rps and its resistance signaling component ndr. the biogenesis of this nat-sirna is also under the control of dicer-like rdr rna polymerase iva, hyl and hen. this finding indicates that multiple layers of control exist in nat-sirna biogenesis and that the induction of the sense transcript of a nat pair is necessary but may not be sufficient to induce some of the nat-sirnas. our recent studies of stress-induced nat-sirnas demonstrated that the biogenesis of these nat-sirnas also depends on rdr and poliva subunit nrpd1a, the components required for rna replication. thus, dsrna formed by sense-antisense pairing might involve a secondary rna replication loop to form nat-sirnas. this rna amplification step may extend beyond the overlapping region to form sirnas outside the overlapping region. we hypothesize that these nat-sirnas may ensure that the antisense gene is silenced. furthermore, "tiling" microarray studies to assay transcript levels throughout the genomes have revealed that more than 30% of the arabidopsis genome and about 60–80% of mammalian genomes can produce transcripts from both strands. about genes in arabidopsis showed significant antisense rna expression. interestingly, a recent study of rice involving robust-long serial analysis of gene expression revealed that many antisense transcripts were induced after the infection of a fungal pathogen magnaporthe grisea, which induces rice blast disease. therefore, antisense transcription is not only limited to the nats but also applies to many non-nat genes. antisense transcription potentially forms dsrnas, which can be recognized by rnai machinery and generate small rnas to guide gene expression regulation. that antisense transcription can be induced by pathogen infection is of particular interest. we have discovered that many small rnas are also induced by pathogen infection. we are currently exploring whether some of these pathogen-induced small rnas may be generated by the induction of antisense transcription outside the nat regions.  we have performed a comprehensive gene expression analysis of arabidopsis cis-nats and discovered that a significant fraction of the sense and antisense partners of cis-nats are anti-correlated, with inverse changes in expression in response to environmental or developmental cues. we identified nearly unique sirnas that match cis-nat genes. the sirnas are enriched in the overlapping regions of negatively correlated nat pairs, which supports our hypothesis that sirna-mediated gene silencing may be one of the important mechanisms in gene regulation of some nat genes. 
the hallmarks of rnai regulation of nats are 1) inverse regulation of two genes in a cis-nat pair by environmental and developmental cues and 2) generation of sirnas by cis-nat genes. in spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts. the negatively correlated expression patterns of sense and antisense genes as well as the presence of sirnas in many of the cis-nats suggest that sirna regulation of cis-nats via the rnai pathway is an important gene regulatory mechanism for at least a subgroup of cis-nats in arabidopsis. however, the extent of sirna-mediated regulation of cis-nat genes is still unclear in any genome. a subset of the cis-nat genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. by searching the arabidopsis small rna project and massively parallel signature sequencing small rna databases as well as our stress-treated small rna dataset, we found small rnas that matched at least one gene in pairs out of protein-coding cis-nat pairs, which suggests that sirnas may regulate the expression of many cis-nat genes. we examined arabidopsis transcript profiling data from public microarray databases to identify cis-nat pairs whose sense and antisense transcripts show opposite expression changes. furthermore, the majority of the putative sirnas within the overlapping regions tend to target only one transcript of a given nat pair, which is consistent with our previous finding on salt- and bacteria-induced nat-sirnas. experimental evidence for the regulation of two cis-nat gene pairs by natural antisense transcripts-generated small interfering rnas via the rna interference pathway has been reported in arabidopsis. putative sirnas have the potential to target more than one gene and half of these small rnas could target multiple members of a gene family. in addition, we found that genes encoding plastid- or mitochondrion-targeted proteins are over-represented in the arabidopsis cis-nats and that 19% of sense and antisense partner genes of cis-nats share at least one common gene ontology term, which suggests that they encode proteins with possible functional connection. the widespread occurrence of cis-nats suggests an evolutionary advantage for this type of genomic arrangement.
 in spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts. the widespread occurrence of cis-nats suggests an evolutionary advantage for this type of genomic arrangement. experimental evidence for the regulation of two cis-nat gene pairs by natural antisense transcripts-generated small interfering rnas via the rna interference pathway has been reported in arabidopsis. however, the extent of sirna-mediated regulation of cis-nat genes is still unclear in any genome.  the hallmarks of rnai regulation of nats are 1) inverse regulation of two genes in a cis-nat pair by environmental and developmental cues and 2) generation of sirnas by cis-nat genes. we examined arabidopsis transcript profiling data from public microarray databases to identify cis-nat pairs whose sense and antisense transcripts show opposite expression changes. a subset of the cis-nat genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. by searching the arabidopsis small rna project and massively parallel signature sequencing small rna databases as well as our stress-treated small rna dataset, we found small rnas that matched at least one gene in pairs out of protein-coding cis-nat pairs, which suggests that sirnas may regulate the expression of many cis-nat genes. putative sirnas have the potential to target more than one gene and half of these small rnas could target multiple members of a gene family. furthermore, the majority of the putative sirnas within the overlapping regions tend to target only one transcript of a given nat pair, which is consistent with our previous finding on salt- and bacteria-induced nat-sirnas. in addition, we found that genes encoding plastid- or mitochondrion-targeted proteins are over-represented in the arabidopsis cis-nats and that 19% of sense and antisense partner genes of cis-nats share at least one common gene ontology term, which suggests that they encode proteins with possible functional connection.  the negatively correlated expression patterns of sense and antisense genes as well as the presence of sirnas in many of the cis-nats suggest that sirna regulation of cis-nats via the rnai pathway is an important gene regulatory mechanism for at least a subgroup of cis-nats in arabidopsis. 