- Research Article
- Open Access
First insights into the nature and evolution of antisense transcription in nematodes
- Christian Rödelsperger†1Email author,
- Kevin Menden†1, 2,
- Vahan Serobyan1,
- Hanh Witte1 and
- Praveen Baskaran1
© The Author(s) 2016
- Received: 14 March 2016
- Accepted: 11 August 2016
- Published: 22 August 2016
The development of multicellular organisms is coordinated by various gene regulatory mechanisms that ensure correct spatio-temporal patterns of gene expression. Recently, the role of antisense transcription in gene regulation has moved into focus of research. To characterize genome-wide patterns of antisense transcription and to study their evolutionary conservation, we sequenced a strand-specific RNA-seq library of the nematode Pristionchus pacificus.
We identified 1112 antisense configurations of which the largest group represents 465 antisense transcripts (ASTs) that are fully embedded in introns of their host genes. We find that most ASTs show homology to protein-coding genes and are overrepresented in proteomic data. Together with the finding, that expression levels of ASTs and host genes are uncorrelated, this indicates that most ASTs in P. pacificus do not represent non-coding RNAs and do not exhibit regulatory functions on their host genes. We studied the evolution of antisense gene pairs across 20 nematode genomes, showing that the majority of pairs is lineage-specific and even the highly conserved vps-4, ddx-27, and sel-2 loci show abundant structural changes including duplications, deletions, intron gains and loss of antisense transcription. In contrast, host genes in general, are remarkably conserved and encode exceptionally long introns leading to unusually large blocks of conserved synteny.
Our study has shown that in P. pacificus antisense transcription as such does not define non-coding RNAs but is rather a feature of highly conserved genes with long introns. We hypothesize that the presence of regulatory elements imposes evolutionary constraint on the intron length, but simultaneously, their large size makes them a likely target for translocation of genomic elements including protein-coding genes that eventually end up as ASTs.
- Conserved synteny
- Non-coding RNA
- Long introns
- Regulatory element
Eukaryotic genomes are packed with regulatory arsenals ranging from transcription factors, histone and DNA modifications, enhancers and silencers, to microRNAs and many other non-coding RNA species. Numerous findings that small alterations in any of these mechanisms can cause rather dramatic developmental defects [1–4], support the idea that these complex regulatory mechanisms ensure the correct spatio-temporal expression patterns that are needed to convert a fertilized egg to a fully grown specimen of its kind. Recently, another species of RNAs, so called antisense transcripts (ASTs), came into focus of research in gene regulation ([5, 6], see  for review). Antisense transcripts are RNA molecules that are transcribed from the opposite strand of a protein-coding gene. While many of reported ASTs are non-coding RNAs, they can also have protein-coding capacity. Several mechanisms have been discovered by which ASTs can affect the transcription of their sense counterpart in practically every transcriptional state. The transcription initiation of genes can be affected by antisense transcripts through modification of methylation states , recruitment of Polycomb proteins  or even histone modifications . Antisense transcription can also affect genes co-transcriptionally by e.g. polymerase collision, which can occur when genes on opposite strands are transcribed simultaneously . Isoform production of a sense mRNA can furthermore be influenced by ASTs if they bind to specific splice sites . Even post-transcriptional effects can be observed as ASTs can stabilize mRNAs by binding to specific sites of the mRNA that would otherwise lead to degradation .
In this study, we characterize genome-wide patterns of antisense transcription in the nematode Pristionchus pacifcus and investigate their evolutionary conservation. P. pacificus has been established as a satellite model to Caenorhabditis elegans for comparative studies of various aspects of evolutionary biology including developmental plasticity [14, 15], immunity [16, 17], and population genomics [18, 19]. In contrast to many other studies, that explicitly aimed to identify and study non-coding RNAs [20–22], our focus is on describing antisense transcription in general, irrespective of protein-coding or non-coding potential. We sequenced a strand-specific RNA-seq library to identify ASTs and to characterize their corresponding host genes. We show that in contrast to the prominent role ASTs play in gene regulation , most identified ASTs in P. pacificus are protein-coding and do not seem to have any regulatory function with respect to their host gene. Nevertheless, we find antisense gene pairs that seem to be remarkably conserved across the nematode phylum. However, even in these highly conserved cases, we see abundant structural changes ranging from intron gain to loss of ASTs. In summary, there seems to be no obvious functional relationship beetwen ASTs and their host genes in nematodes. Antisense transcription rather appears to be a feature of highly conserved genes with exceptionally long introns. What imposes the evolutionary constraint on these long introns can only be speculated and will be subject to further studies.
Identification of antisense transcripts in P. pacificus
Majority of antisense transcripts are protein-coding
We first tested if ASTs show any signal for non-coding RNAs. We therefore hypothesized that they should be depleted from homology to any protein-coding gene or proteomic data [24, 25]. To test this, we compared the amount of protein evidence for antisense transcripts with sets of randomly chosen transcripts having the same length distribution. In contrast to our expectation that ASTs should be depleted in protein evidence, they actually exhibitted elevated levels (compared to randomly chosen transcripts) of BLASTX hits in correct orientation against available proteomic data (Fig. 1 c) and predicted protein-coding genes (Fig. 1 d). The different protein-coding potential between ASTs and randomly chosen transcripts is probably due to differences in proportions of non-coding RNAs, protein-coding genes, but also mis-annotated transcripts. However, the main conclusion from this analysis is that ASTs do not show a significant depletion of protein evidence suggesting that they represent to a large extend protein-coding genes. Consequently, this indicates that antisense transcription in general is a poor predictor of non-coding RNAs. Consistently, a previous study that screened for non-coding RNAs in C. elegans identified only 60 candidates for antisense non-coding RNAs . As ASTs tend to be protein-coding, we used the annotation of protein-coding genes from C. elegans to identify an analogous set of 467 ASTs embedded in 362 host genes in the C. elegans genome for further analysis.
Uncorrelated expression profiles between ASTs and host genes
Even though most ASTs do not seem to represent non-coding RNAs, we tested whether ASTs could have any regulatory impact onto their host genes or vice versa. As antisense transcription has been reported to affect gene expression by polymerase collision , we tested whether gene pairs of host genes and their ASTs are more likely to show correlated or anticorrelated expression than random gene pairs. We obtained developmental expression profiles from P. pacificus  and large-scale expression profiles for C. elegans (see “Methods”), and compared the distribution of Spearman correlation coefficients between expression levels of antisense gene pairs. Based on this analysis, we find that antisense gene pairs do not show any non-random signal towards correlated nor anticorrelated expression (Fig. 1 e, f). This suggests that expression of ASTs and host genes are controlled by independent regulatory programs with no evidence of interference, which is in contrast to studies in mammals where coexpression of antisense gene pairs has been found [5, 6].
Conserved gene pairs represent unusually large blocks of conserved synteny
To assess the degree of conservation of antisense gene pairs, we used TBLASTN searches and orthology predictions between P. pacificus and C. elegans  to test whether orthologous host genes harbor the same antisense transcripts in both species. Indeed, we initially found 13 cases where antisense gene pairs are clearly conserved between C. elegans and P. pacificus (Fig. 1 g, h). In addition, comparing the size of the spanned conserved syntenic blocks with all syntenic blocks between C. elegans and P. pacificus, we find that antisense gene pairs in C. elegans span rather large genomic regions as compared to conserved syntenic blocks in general (P=0.03, Wilcoxon test). A similar trend can also be observed in P. pacificus, yet the comparison is not significant (P=0.11, Wilcoxon test). As some conserved gene pairs were probably missed due to the incomplete assembly of host genes from poly-A enriched RNA-seq data, the statistical power would therefore be increased with more complete transcriptome data. The observation of unusually large blocks of conserved synteny does not imply that antisense gene pairs are in any way more conserved than expected, as conservation on the integrity of the host gene alone coupled with a larger gene size (see below) could result in the same pattern.
Limited conservation of antisense transcription in nematodes
Gene structure variation in the highly conserved vps-4/npr-23 pair
We mapped the C. elegans protein sequences of the most highly conserved pair vps-4/npr-23 against 19 nematode genomes and reconstructed gene trees for both genes (Fig. 3). As expected, phylogenetic reconstructions of both gene trees (Fig. 3 a, b) show good agreement with the species tree and major nematode clades are robustly separated [31, 32]. Together with the synteny information (Fig. 3 c) and the fact that BLASTP searches against the NCBI nr database could not identify any inparalogs, this confirms orthology for vps-4 and npr-23 across all analyzed genomes. At the level of gene structures a number of conserved as well as divergent patterns are visible. npr-23 resides in all species in the first conserved intron of vps-4, which happens to be the largest intron. While the relative position of npr-23 within this intron of vps-4 is highly conserved, the gene structure of npr-23 shows independent gains of introns in the lineages leading to clade IV nematodes and to the Pristionchus lineage (Fig. 3 c).
Gene gain and loss in the ddx-27 locus
Lineage-specific loss of antisense transcription
Host genes delineate a highly conserved, but heterogenous gene class with long introns
The above mentioned examples show that ASTs can exhibit many different kinds of structural changes while being localized in large introns of their host genes. At the same time, the host gene is broadly conserved across the nematode phylum. The latter statement is supported by the finding that around 60 % of C. elegans host genes have one-to-one orthologs in P. pacificus, which is much more than compared to 29 % for all C. elegans genes (P<10−16, Fisher’s exact test) . In strong contrast, only 16 % of C. elegans ASTs have one-to-one orthologs in P. pacificus (P<10−16, Fisher’s exact test). Despite their high degree of conservation, host genes represent a functionally heterogenous class of genes, as Gene Ontology term enrichment analysis only detects few and not highly-significant overrepresentations (ATP binding (GO:0005524) and larval development (GO:0002164), P<10−3, ). At the level of protein domains, we found some gene families with DNA binding domains such as CCCH-type (PF00642, P<10−8, Fisher’s exact test with FDR correction) and PHD-finger zinc fingers (PF00628, P<10−6), as well as BTB domain containing genes (PF00651, P<10−8), but also ATP synthases (PF00895, P<10−6), phosphotransferases (PF01636, P<10−5), deoxyribonucleases (PF03265, P<10−5) and amino acid permeases (PF00324, P<10−5).
Little non-coding conservation between P. pacificus and C. elegans
The easiest explanation that would explain the conservation of large introns is the presence of important regulatory sequences. Such a setting would resemble the finding of genomic regulatory blocks in vertebrates that link regulatory elements with conserved synteny around developmental genes [37, 38]. To test whether introns of host genes harbor conserved regulatory elements, we performed BLASTN searches between non-coding/non-repetitive stretches of the P. pacificus and C. elegans genomes. In strong contrast to analyses in vertebrates that identified thousands of highly conserved non-coding elements between human and zebrafish [37, 39], we only found 28 non-coding and non-repetitive sequences in P. pacificus with recognizable homology at the nucleotide level to 27 sequences in C. elegans (BLASTN e-value <0.001). We thus speculate that long introns of host genes harbor regulatory elements, but these elements evolve faster than the protein products of their targets, which is in contrast to studies in vertebrates that show abundant conservation at non-coding and protein level [37, 39].
In the last couple of years an increasing number of studies have demonstrated evidence for important regulatory roles of antisense transcription in various organisms ([5, 6, 40] see  for review). To characterize antisense transcription in the nematode Pristionchus pacificus, we sequenced a strand-specific RNA-seq library and identified 1112 configurations of antisense transcription. Classifying these cases showed that the majority represents ASTs that are fully embedded in introns of their host genes. In contrast to the presumed regulatory role as suggested by studies in yeast, humans and plants that identified patterns of coexpression as well as mutual exclusiveness [5, 6, 40], P. pacificus ASTs preferentially encode proteins and their expression is completely uncorrelated to the expression of their host genes. This suggests that there is no obvious regulatory interaction between ASTs and host genes. As it is difficult to compare studies that were based on different data sets, and methods, we would like to point out that the only study by Sun et al. which employed a common protocol for multiple organisms demonstrated an exceptional role of antisense transcription in nematodes . More precisely, Sun et al. found that compared to several vertebrates and insects, C. elegans had the lowest proportion of antisense gene pairs as a fraction of all genes  but unfortunaley they did not test for their regulatory function. Despite the lack of obvious regulatory interactions between ASTs and their host genes in P. pacificus, we found a few cases that showed patterns of broad conservation across the nematode phylum, with the gene pair vps-4 and npr-23 representing the only gene pair that is conserved in all twenty nematode genomes. This could be indicative of some other kind of constraint acting on preservation of such gene configurations. As it is not possible to distinguish constraint acting on the host gene alone from evolutionary constraint preserving the antisense configuration, it is extremely difficult to assess whether the observed conservation is greater than expected. We therefore chose to gain further insights into the evolution of antisense gene pairs by investigating the most highly conserved cases in greater detail. We found that ASTs can undergo a variety of structural changes such as gene loss, translocations, and intron gain. The small degree of conserved association between host genes and ASTs let us hypothesize that antisense transcription is a feature of highly conserved genes with long introns. This could potentially explain, why a recent study that screened for non-coding RNAs in C. elegans explicitly ignored the top 1 % of genes with longest introns . Such introns do not harbor any highly conserved non-coding elements that are recognizable by nucleotide level sequence identity between C. elegans and P. pacificus, which is in contrast to vertebrates where highly conserved elements can be found between humans and zebrafish . Similarly, in contrast to studies in vertebrates where conserved non-coding elements are strongly clustered around highly conserved developmental genes [39, 42], host genes in nematodes, despite their evolutionary conservation, represent a very heterogeneous class of genes as is indicated by the lack of strong signals in functional enrichment analyses. Finally, since functionally characterized antisense transcripts represent a diverse class of biological sequences [8–13], which as a whole is only poorly understood, our analysis provides first insights into the nature and evolution of antisense transcription in nematodes, showing that, at least in P. pacificus, transcriptomic signals for antisense transcription are mostly dominated by embedded protein-coding genes. However, we cannot exclude that there are non-coding regulatory antisense transcripts in our P. pacificus data set, of which we only generally characterized the most abundant class. Yet our study demonstrated that they by far do not represent the bulk of ASTs and more rigorous filtering techniques [20, 43] are probably needed to distill a candidate set of antisense non-coding RNAs. The most interesting question remains what imposes the conservation on long introns in nematodes. It is tempting to speculate that they harbor regulatory elements such as enhancers and silencers or small RNAs, but even if this was true, our analysis has shown that these non-coding regulatory elements evolve so fast that they are not recognizable by sequence homology between C. elegans and P. pacificus, which stands in contrast to the conservation of protein sequences of their respective host genes. This explanation would be in accordance with Davidson’s theory on regulatory network evolution stating that while mutations in pleiotropic genes likely affect all their functions throughout development and will probably have quite severe effects, mutations in individual cis-regulatory modules will only affect one particular phase of activity and can therefore more easily be tolerated . However further analysis at much closer evolutionary distance [45–47] would be needed to identify and characterize such regulatory networks in further detail.
Our analysis shows that antisense transcription is a feature of highly conserved genes with long introns and ASTs tend to be less conserved and protein-coding genes that do not show any regulatory effect on their respective host genes. The large size and little sequence conservation of long host gene introns makes them an ideal target for translocation of other genomic elements including protein-coding genes that eventually end up as ASTs. Our results may help to guide other studies in different taxonomic groups and help to gain an unbiased view of antisense transcription.
Worm cultures and RNA-seq experiment
The P. pacificus reference strain PS312 (California) was kept on 6-cm plates with nematode growth medium (NGM) agar and was fed with a lawn of Escherichia coli OP50 grown in 400 μl L-Broth. Cultures were maintained at 20 ° C. Culture populations were allowed to grow until their food was exhausted, immediately after which the cultures were processed for sequencing. Five mixed-stage plates were washed with 40 ml M9, centrifuged immediately at 1300 g for 4 min, rinsed with 40 ml 0.9 % NaCl treated with 40 μl ampicillin and 40 μl chloramphenicol and shaken gently for 2 h, and finally concentrated into a pellet by centrifugation and immediately frozen in liquid nitrogen. NEBNext Ultradirectional RNA Library Kit (Cat # E7420L) was used to prepare libraries. RNA-Seq libraries were sequenced as 2 ×101-bp paired-end reads on an Illumina HiSeq 2000, yielding 30.6 million reads. Raw reads were submitted to the NCBI sequence read archive (accession SRX1609204).
Identification of antisense transcripts in P. pacificus and C. elegans
Raw reads were aligned to the P. pacificus genome assembly (version Hybrid2) by means of the software TopHat (version v2.0.14)  with default settings. The P. pacificus Hybrid2 assembly represents and improved version of the Hybrid1 genome assembly  that was generated by merging scaffolds and filling gaps in the assembly based on PacBio sequencing data of the reference strain (PS312) and with the help of the software PBJelly2 and GapCloser of the SOAP suite [49, 50]. This reduced the number of scaffolds from 18,083 to 12,395 and increased the N50 value from 1.2 to 1.4 Mb. We then assembled 42,821 transcripts using the reference-guided transcriptome assembler cufflinks (version v2.0.1) . ASTs were defined as assembled transcripts that completely fall into non-exonic regions of a transcript on the opposite strand. Coordinate overlaps between transcripts from both strands were done using bedtools (version 2.17.0) . Figure 1 b shows the distribution of various classes of overlaps.
To define ASTs in C. elegans, we relied on the available gene annotations from Wormbase (release WS140). This yielded 467 ASTs and 362 host genes. One-to-one orthology predictions between C. elegans and P. pacificus were generated by best-reciprocal BLASTP searches including inparalog filtering [19, 26]. To compare the degree of conservation of antisense gene pairs with blocks of conserved synteny between C. elegans and P. pacificus, we ran the software CYNTENATOR  with the following parameter settings: -gap –0.5 -coverage 1 -thr 3 -length 2, to compute a baseline distribution of conserved gene orders between both species.
Enrichment test for protein-coding genes
To test for evidence of translation and coding potential in ASTs, we employed proteome data from two previous studies [24, 25] and P. pacificus gene predictions. We then tested, to what extent ASTs show BLASTX hits in the corresponding protein databases and compared these numbers to sets of randomly chosen transcripts with a similar size distribution, i.e. ASTs were divided based on their length into 20 sets holding each 5 % of ASTs. For each set that corresponded to a specific length range, a number of transcripts equivalent to the number of ASTs in the respective set was randomly chosen from all transcripts in the corresponding length range. The random sampling was repeated 1000 times to generate a baseline distribution of expected number of BLASTX hits. As our transcriptome data has directional information, we filtered out BLASTX hits that referred to the reverse strand of the query transcripts.
Analysis of various expression profiles
We downloaded expression-profiling data throughout the development of P. pacificus . For C. elegans, 30 expression profiles from different developmental stages including embryo, larvae, and adults  as well as 20 expression profiles of mutants and different environmental conditions were downloaded. These data sets were taken to test whether antisense gene pairs are more likely to show either correlated or anticorrelated expression patterns than randomly chosen gene pairs (Fig. 1 e, f).
Identification of highly conserved antisense gene pairs
To screen for conserved antisense gene pairs, we defined protein sequences of antisense gene pairs from C. elegans as queries for TBLASTN searches (version 2.2.28+) against 20 nematode genomes. These genomes were retrieved from WormBase and pristionchus.org: Ancylostoma ceylanicum (WS248), Ascaris suum (WS240), Brugia malayi (WS240), Bursaphelenchelus xylophilus (WS240), Caenorhabditis angaria (WS240), Caenorhabditis briggsae (WS240), Caenorhabditis elegans (WS240), Caenorhabditis remanei (WS240), Dirofilaria immitis (WS240), Heterorhabditis bacteriophora (WS240), Haemonchus contortus (WS240), Loa loa (WS240), Meloidogyne hapla (WS240), M. incognita (WS240), Necator americanus (WS248), Onchocerca volvulus (WS250), Panagrellus redivivus (WS240), Trichinella spiralis (WS240), P. pacificus (HYBRID2), and P. exspectatus (ALLPATH-LG) . For each target contig and strand, leftmost and right most positions TBLASTN hits (e–value <0.001) for host gene and AST were determined and finally checked, whether the spanned region for the host gene hits contains the corresponding AST hits on the opposite strand (Fig. 2). To further confirm these results in individual cases, query sequences from C. elegans were mapped using exonerate (version 2.2.0) against the target genome (Figs. 3 c, 4 c and 5 c). In individual cases we completed partial hits by using exonerate alignments of sequences with the help of more closely related species (e.g. taking the homologous regions of a closely related species as query instead of the C. elegans sequence).
Protein sequences for all 20 nematodes were extracted based on the exonerate alignments of the C. elegans queries. In a few cases protein sequences were manually completed using protein sequence data from available closely related species (WormBase, pristionchus.org, Genbank). We searched for paralogous genes and non-nematode orthologs in the NCBI nr database and generated multiple sequence alignments using MUSCLE (version 3.8.31) . After model testing with the ProtTest webserver , the best model was then chosen to reconstruct a final Maximum-likelihood tree with bootstrap support values using the R package phangorn . Orthologous sequences of non-nematode species (mostly from arthropods) were used to infer the root of individual trees. We searched for paralogs for given genes in the NCBI nr database but ignored near identical sequences, as it is difficult to decide whether these sequences represent different isoforms, assembly artifacts (e.g. different alleles were called as individual genes), or truly represent very recent duplications.
Identification of highly conserved non-coding elements
Based on the repeat masked genome assemblies of C. elegans and P. pacificus, reciprocal BLASTN searches between non-coding and non-repetitve genomic regions were carried out. The repeatmasked C. elegans genome was downloaded from Wormbase (release WS240). For P. pacificus, repeats were modeled using RepeatModeler and masked using Repeatmasker. In both genomes, we masked any sequence that was annotated as protein-coding. The remaining regions with minimal length of 100 bp were then used as queries for BLASTN searches to find conserved non-coding elements, that we defined as BLASTN hits with e-value <0.001.
We thank Dr. Kay Nieselt for helpful discussions. We thank Barbara Meyer for providing P. pacificus PacBio data and Erich Schwarz for improving the P. pacificus assembly.
This work was funded by the Max Planck Society.
Availability of data and materials
Raw reads have been submitted to the NCBI short read archive under the study accession SRX1609204. Genome and annotations are available at http://www.pristionchus.org . Sequences, alignments, and trees have been submitted to datadryad.org and are accessible at doi:10.5061/dryad.4gd87.
CR conceived and supervised the study. VS and HW carried out the experiments. KM, PB, and CR analyzed the data and wrote the manuscript. All authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study does not involve research on humans or human material and also not on animals according to the german animal protection legislation. Therefore no ethical approval is needed.
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