BACKGROUND
transcriptome plasticity is a powerful modulator of gene expression that provides multicellular organisms with the complexity needed to drive development and maintain tissue identity.
apart from a few cases, we still do not fully understand what specific transcriptome rearrangements occur in somatic tissues, and how they integrate with gene regulation at the post-transcriptional level to maintain tissue and cellular diversity.
the small nematode caenorhabditis elegans is an ideal model organism to study these events, since its gene model has been extensively characterized in past years  <cit> .
it is also experimentally tractable with short and precise developmental timing, approximately  <dig>  somatic cells, a transparent and simple body plan and an entirely defined cell lineage  <cit> .
large-scale efforts have detailed its transcriptome at a global level  <cit> .
promoter diversity  <cit> , alternate splicing events  <cit>  and changes in 3’ untranslated regions   <cit>  are also well characterized.
while the formation of several worm tissues and the genes involved in driving these processes have been extensively described  <cit> , we still do not fully understand how the synergistic activity of tissue-specific events before, during and after transcription drive and maintain tissue identity.
pre-transcriptionally, enrichment of sequence-specific elements within c. elegans promoters has been linked to tissue-specific changes in gene expression  <cit> , suggesting that these elements, together with trans-acting factors that recognize them, are fundamental for driving the transcriptional programs of unique tissues.
during transcription, mrna isoforms resulting from alternative splicing increase transcriptome complexity, coordinating tissue development  <cit> .
recently, a genome-wide study in c. elegans found that thousands of transcripts are alternatively spliced and many of them change splicing patterns during development  <cit> , suggesting that tissue-specific splicing may play key roles in this process.
post-transcriptionally, 3’utrs are known to contain multiple regulatory sequence elements important for gene regulation  <cit> .
recently, two independent studies suggest that more than 40% of worm genes possess 3’utrs subjected to alternative polyadenylation , a mechanism that generates multiple 3’utr isoforms for the same genes  <cit> .
this process is widespread in metazoans  <cit> , coordinated through development  <cit> , and misregulated in disease  <cit> , underscoring a potential role for apa in tissue-specific modulation of gene expression.
the cleavage and polyadenylation of nascent mrnas in eukaryotes is mainly executed by two large multimeric complexes named cleavage and polyadenylation specificity factor  and cleavage stimulation factor   <cit> .
cpsf recognizes and binds to the polyadenylation ) signal  element located approximately  <dig> nt from the polya site in the 3’utr of mrnas.
in metazoans, the pas sequence is commonly aauaaa  <cit> .
this sequence is necessary and sufficient for 3’end polyadenylation  <cit> .
cstf directly interacts with cpsf and binds to gu-rich elements downstream of the cleavage site  <cit> .
although apa is pervasive in worms and correlated with development, suggesting that apa functions in worms tissues  <cit> , it is unclear whether apa is tissue-specific.
both cpsf and cstf are likely to have a role in managing the choice between pas elements in the same 3’utr and inducing apa.
there may also be additional tissue-specific accessory factors that modify the basal polyadenylation machinery, controlling the usage of one pas element over another.
tissue-specific isoforms of the cpsf or cstf complexes could be responsible for apa  <cit> .
over a decade ago, stoichiometric levels of cstf members were indeed shown to control apa in b cell activation  <cit> , and recent high-throughput approaches showed that other factors might also play important roles in modulating apa  <cit> .
other processing factors were also recently shown to influence the location of cleavage  <cit> .
these studies underscore the importance of the correct stoichiometric ratio of each of the 3’end processing factors for producing a mature mrna.
surprisingly, it was also recently shown that u <dig> snrnp is involved in this process, suggesting possible cross talk between apa and the rna splicing machinery  <cit> .
these models may not be mutually exclusive.
in c. elegans, the isolation of tissue-specific mrna to study transcriptome plasticity and apa is challenging due to the lack of in vitro cell cultures, the worm’s tough outer cuticle that interferes with sample preparation and the small size of many tissues that prevents manual dissection.
several techniques have been developed to circumvent these issues, including fluorescence-activated cell and nuclear sorting  <cit> , nuclei-tagging  <cit>  and mrna-tagging  <cit> .
in particular, mrna-tagging has been widely used to isolate and study mrna from muscle  <cit> , epithelial  <cit> , hypodermal  <cit> , neuronal  <cit>  and seam cells  <cit> .
this technique uses tissue-specific promoters to drive expression of a flag epitope-tagged cytoplasmic poly-a binding protein , which specifically binds to the poly-tail of mrnas in the cytoplasm, followed by crosslinking and immunoprecipitation of tissue-specific mrnas.
thus far, mrna-tagging has been coupled with dna microarrays and genomic tiling arrays, which both lack the advanced sensitivity and specificity possible today with deep sequencing, potentially limiting the results obtained with this methodology.
recently, a novel method that couples tissue-specific nuclei isolation with deep sequencing was used to analyze the c. elegans intestine transcriptome  <cit> .
unfortunately, while enhancing the sensitivity of tissue-specific mrna extraction and sequencing, this technique limits the analysis to nuclear mrna species, and only identifies short 3’end portions of 3’utrs that need to be bioinformatically attached to their closest genes, potentially introducing mapping inaccuracies.
in addition, this method does not provide tissue-specific mrna isoform data, losing an important component needed to study the transcriptome plasticity of individual tissues.
integrating mrna-tagging with rna-seq analysis could significantly improve the resolution of these studies and identify additional factors controlling tissue development and identity.
here, we have improved tissue-specific transcriptome profiling in c. elegans, optimizing mrna-tagging for deep sequencing.
we call this updated method polya-tagging and sequencing .
we have applied pat-seq and profiled tissue-specific mrna from the c. elegans intestine and two muscle tissues belonging to the pharynx and body wall of mixed stage worms.
we describe and compare gene expression, promoter sequence composition, mrna isoforms changes and alternative polyadenylation between each tissue.
pat-seq significantly improved the resolution of tissue-specific transcriptomes from previous studies, adding thousands of novel genes and isoforms that allow for a more comprehensive analysis of them.
in addition, we have used pat-seq to profile the transcriptome of a previously uncharacterized tissue , allowing us for the first time to directly compare gene expression changes between different tissues from the same organism at a higher resolution, using the same experimental settings.
we find that transcript diversity detected among these three tissues is mirrored by the presence of characteristic tissue-specific promoter signatures.
in addition, we found that apa is widely used at a tissue-specific level, highlighting major complex tissue-specific transcript dynamics and post-transcriptional regulatory mechanisms.
we describe a large number of 3’utr isoforms specifically expressed in each tissue and find that these 3’utrs are enriched for experimentally and bioinformatically predicted microrna  targets, suggesting that tissue-specific apa is used in worms as a mechanism to interface with mirna mediated post-transcriptional gene regulation.
finally, we have remapped, incorporated and curated 3’utr data from previously published studies  <cit>  and integrated these data with our new tissue-specific datasets.
the database is accessible through our new worm apa-specific website  <cit> , which is publically available and represents a unique resource for the scientists interested in 3’utr biology.
RESULTS
isolation of mrnas from intestine and muscle tissues
mrna-tagging has so far been coupled with low-resolution platforms, such as microarrays and tiling arrays, that lack the sensitivity required to detect low expressed transcripts, pinpoint gene isoform changes and map 3’utrs at single base resolution.
to improve upon its sensitivity and specificity, we made several key changes to the original mrna-tagging protocol  <cit> : 1) we added three tandem flag-epitope  tags instead of one, to improve the efficiency of the flag pull-down  <cit> .
2) in the original mrna-tagging protocol, the flag-tagged pabpc construct is selectively expressed as extra chromosomal arrays, which are unstable and often lead to mosaic expression patterns, or integrated as multiple copy lines  <cit> .
we instead opted for the widely used mos- <dig> single copy insertion  technology  <cit> , which stably incorporates the construct of interest in the worm genome.
3) we adopted a novel strategy to prepare the tissue-specific cdna libraries that relies on linear amplification of mrnas, minimizing the quantification error rate due to limited starting material, providing high-quality transcriptome and 3’end data in the same experiment  <cit> ; and 4) replaced the microarray step with next generation sequencing  to improve data resolution.
we used tissue-specific promoters to drive the expression of the c. elegans cytoplasmic pabpc gene , in-frame with gfp and fused to a 3 × flag tag , in intestine, pharynx or body muscle .
as mossci technology mediates transgene and rescue cassette  insertion into a specified region of chromosome ii, we confirmed this integration event in each transgenic worm line  and verified its expression using western blotting .figure  <dig>
pat-seq of intestine and muscles tissues.
pat-seq uses gateway-compatible  entry vectors expressing the polya-pull cassette in each tissue using tissue-specific  promoters.
polya-pull expressed in the intestine , pharynx , and body muscle .
expression of polya-pull produces a 3 × flag-tag  fused to pab- <dig> , which specifically binds to the poly tails of mrnas .
the complex is immunoprecipitated using α-flag beads.
tissue-specific cdna libraries are sequenced and mapped onto the ws <dig> gene model.
detection of stable integration of the polya-pull cassette.
left panel: using pcr we detected genomic integration of the common portion of the polya-pull cassette  in each tissue.
the negative control, myo-2Δpab- <dig>  was also integrated.
right panel: western blotting using α-flag antibodies detected the in-frame polya-pull fusion protein in lysates from transgenic worms expressing it in the pharynx  but not in lysate from wild type n <dig> worms.
quantification of the specificity and sensitivity of the pull-down using rt-pcr: left panel: myo- <dig>  , ges- <dig>   and dpy- <dig>  transcripts were detected in total rna extracted from wild type n <dig> worms.
middle panel: using immunoprecipitation, we successfully detected the presence of the muscle-specific gene myo- <dig>   and the exogenous unc- <dig> 3’utr , but not the intestine-specific ges- <dig>   and the hypodermis-specific dpy- <dig> .
these transgenic worms expressed polya-pull cassette in the pharynx, but not in our negative control in wild type n <dig> worms .
the primers used to detect unc- <dig> 3’utr also detected 18s rrnas .
this band was replaced with two unc- <dig> 3’utr isoforms , suggesting that polya-pull enriched for polya+ rnas.
right panel: we are unable to isolate tissue-specific rna from worms lacking pab- <dig> .
we tested the sensitivity and tissue-specificity of our mrna pull-down approach using worms expressing polya-pull in the pharynx  , validating the ability of our construct to selectively bind muscle specific transcripts .
the known intestine-specific transcript ges- <dig>  and hypodermis-specific dpy- <dig>  were depleted from the same sample.
immunoprecipitation from wild type n <dig> worms yielded no detectable background in rt-pcr for the same genes .
to test if our polya-pull construct selectively binds polya+ rnas, we prepared a gfp::3 × flag fusion protein that does not contain pab- <dig>  this vector is unable to bind polyadenylated mrnas .
we expressed this new construct in the pharynx .
as expected, using this construct, we were unable to detect the pharynx-specific myo- <dig> transcript .
taken together, these results suggest that our polya-pull construct effectively enriches tissue-specific mrnas and specifically binds polya+ rnas.
pat-seq analysis of mrnas from intestine and muscle tissues
we then prepared our tissue-specific libraries with two biological replicates .
we also performed two negative control pull-down experiments expressing the Δpab-1-pull construct in the pharynx to optimize the sensitivity and specificity of our approach.
following the pull-down, the cdna libraries were prepared using isothermal linear cdna amplifications, which allows cdna synthesis across the full length of the transcripts with as little as  <dig> ng of total rna, thus improving the coverage of our whole-transcriptome amplification independently of the 3’polya tail  <cit> .
we, then, barcoded, pooled and sequenced our eight rna pull-down libraries on the illumina hiseq- <dig> platform .
the resulting paired reads were computationally assembled and mapped onto the c. elegans ws <dig> genome.
a summary of the results of this mapping is displayed in table s <dig> in additional file  <dig>
we obtained approximately  <dig> m unique reads per sample .
the number of genes mapped in each tissue was consistent between biological replicates, reflecting the robustness of our library preparation from tissue-specific rna samples.
the overall number of genes and their ratio detected in each biological replicate, with the exception of our negative control, was comparable  , suggesting that our approach was consistent.
in the intestine, we detected a much larger number of genes  compared with that of pharynx  and body muscle  tissues.
genes and their expression levels between biological replicates correlated well, with the exception of the myo2p::Δpab- <dig> control, further supporting the reproducibility of our approach and suggesting that pat-seq is specific and sensitive .
a closer look into transcripts recovered with myo2p::Δpab- <dig> revealed an enrichment of ncrnas and other common contaminants, reinforcing that this sample represented random non-specific rna pulled-down during the immunoprecipitation step .
we have validated the tissue localization of selected tissue-specific genes identified with pat-seq by cloning their promoters and using them to drive expression of gfp in vivo .
we detected a common core set of approximately  <dig>  unique genes present in all three tissues.
these transcripts include housekeeping genes, such as actin, histone genes, ribosomal proteins, genes involved in transcription, basal transcription factors, and genes involved in dna maintenance and replication.
a complete list of genes and isoforms detected in each tissue is shown as a diagram in figure  <dig>  top panel and comprehensively in table s <dig> in additional file  <dig> figure  <dig>
distribution of tissue
-specific gene expression and alternative polyadenylation in intestine,
pharynx and body muscle.
top panel: tissue-specific genes identified by pat-seq and the distribution of their expression levels between each tissue.
a large pool of  <dig>  genes is uniquely expressed in the intestine, while a smaller portion of  <dig> and  <dig> genes is expressed uniquely in the pharynx and body muscle, respectively.
we have detected a common set of  <dig>  genes expressed in all three tissues.
edges represent the presence of transcripts in each tissue, and color-coding indicates expression levels of genes in tissues .
bottom panel: the  <dig>  genes shared in all three tissues were further sorted based on the 3’utr isoform and their expression levels.
approximately 25% to 30% of these genes use common 3’ends in these three tissues, while the remaining 70% use tissue-specific 3’utr isoforms.
the intestine transcriptome is expansive, expressing over 30% of c. elegans mrnas
the intestine is one of the largest tissues in c. elegans, composed of  <dig> large cells and a total of  <dig> to  <dig> nuclei, with a final 32-fold polyploidy in the adult worm.
it is also one of the most functionally diverse tissues, participating in digestion, nutrient transport and storage, innate immunity, response to environmental toxins, defecation and dauer formation  <cit> .
while the intestine transcriptome has been studied extensively  <cit> , our results correlated with and significantly expanded these studies .
we detected a total of  <dig>  expressed genes in this tissue , of which  <dig>  genes and  <dig>  spliced isoforms are uniquely expressed in the intestine, but not in either muscle tissue .
the most abundant in this dataset were metabolic enzymes and nutrient transport genes, consistent with this tissue’s physiological function in digestion .
among these intestine-only genes, we identified  <dig> unique transcripts that contain a dna binding domain and were previously described as transcription factors  <cit>  .
we speculate that these transcription factors may contribute to the gene regulatory network necessary for tissue identity and function.
as expected, members of the gata family are among the most abundant transcription factors.
these factors bind ‘gata’ elements on promoters and have been shown to regulate endoderm specification and all aspects of intestine development and function in c. elegans  <cit> .
in addition, a significant portion  of all transcription factors uniquely expressed in this tissue  are members of the nuclear hormone receptor family.
many members of this class of transcription factors were also previously shown to regulate c. elegans metabolism  <cit> .
we also detected a large pool of novel intestine-specific transcription factors with unknown roles that need to be further investigated .
recently, pauli and colleagues coupled mrna-tagging with dna microarrays from l4-stage worms and identified  <dig>  intestine transcripts  <cit>  .
others produced a sage library of transcripts from dissected worm intestines from mutant adults, and detected a total of  <dig>  intestine genes   <cit> .
a comparison of each of these datasets with ours identified 71% and 78% overlap with mcghee and pauli datasets, respectively , suggesting that the increased sensitivity of pat-seq applied to study the intestine transcriptome of mixed stage worms expanded the core c. elegans intestine transcriptome.
haenni and colleagues  <cit>  recently optimized a procedure for extracting intact nuclei from c. elegans intestine, followed by fluorescence-activated cell sorting  and deep sequencing of mrna, focusing on the 3’ end of the transcriptome.
this approach allowed the authors to map  <dig>  genes expressed in this tissue .
we compared our intestine datasets with these results, downloading and remapping the raw reads from haenni et al.
using the same filtering criteria used in our datasets .
we found that 71% of genes were detected in both datasets, leaving 29% of genes present in the haenni et al.
dataset not present in ours .
despite these differences, the distribution of genes detected in both datasets plotted by expression values correlated .
our inability to detect 1/ <dig> of the genes in haenni et al.
may be attributed to the fundamentally different techniques used in the preparation of our cytoplasmic, intestine-specific mrnas using pat-seq versus nuclear cdna prepared from isolated facs sorted nuclei as in haenni et al.
.
the difference in gene pools may have arisen because of the different cellular origin of the mrnas analyzed by the two studies.
finally, we have overlapped our datasets with mcghee et al.
<cit>  and haenni et al., the two intestine transcriptomes datasets obtained by sequencing, revealing a core set of shared  <dig>  genes .
these  <dig>  genes represent a collection of high confidence intestine expressed genes, supported by at least three independent approaches, and will provide important insights in unraveling the genetic basis of intestine tissue identity in worms.
muscle transcriptomes are smaller but contain mostly unique gene pools
c. elegans possess only two large muscles: the pharynx and the body muscle.
the pharynx, or foregut, is an important developmental model composed of eight layers of muscle, in addition to surrounding neural and epithelial tissue  <cit> .
the muscle component of this tissue is composed of  <dig> cells that coordinate intake and physical crushing of the worm bacterial diet  <cit> , subsequently facilitating raw nutrient transfer to the intestinal lumen for digestion.
while the genetic factors controlling early development of the pharynx have been described in detail  <cit> , individual cell-types of the pharynx are less characterized because genetic signatures belonging to these specific subgroups have not been extensively studied.
we detected  <dig>  genes expressed in pharynx muscle .
among the top genes expressed were several myosin and actin isoforms, and pharynx-specific neurotransmitters, consistent with this tissue’s muscular identity.
importantly, we found only  <dig> unique genes with  <dig> spliced isoforms significantly expressed in this tissue  .
most of these genes have unknown function, with only  <dig> transcripts  described in wormbase so far  <cit> .
the top genes of this list are collagen isoforms and motor protein genes .
within this pool we also detected  <dig> pharynx-specific transcription factors.
their gene targets are mostly unknown .
the body muscle tissue, defined as all non-pharyngeal muscle cells, is homologous to vertebrate skeletal muscle  <cit> , and critical for locomotion, egg laying, defecation, and mating  <cit> .
several groups also studied the transcriptome of this tissue  <cit> .
we detected  <dig>  genes expressed in the body muscle.
similar to our pharynx dataset, within this pool we detected only  <dig> unique genes corresponding to  <dig> spliced isoforms .
the list of top ten genes in the body wall muscle dataset was also enriched for muscle-specific genes such as myosin and actin isoforms .
we also detected a unique gene pool, including previously identified genes, such as those in the calveolin family  <cit> , and type iv collagen  <cit> .
out of predicted worm transcription factors, we identified  <dig> genes that are uniquely expressed in this tissue , including unc- <dig>  a srf-like transcription factor essential for body wall muscle development, and blmp- <dig>  a prdm family member required for embryonic slow muscle fiber formation in vertebrates  <cit> .
using pat-seq we were able to detect many statistically significant isoform expression changes between intestine and muscle tissues .
tissue-specific promoters possess unique signatures
the compact nature of the promoter regions in the c. elegans genome provides us with a unique platform to examine tissue-specific elements in these regions and highlight signatures such as transcription factor binding sites.
we first studied the sequence composition of these promoters, defined as the portion of genomic sequences from - <dig> to + <dig> from the tss of protein coding genes  <cit> .
we compared these sequences to promoters of random genes from the whole c. elegans transcriptome  .
we found that, contrary to higher metazoans, such as in humans where promoters are significantly enriched in cytosines and guanosines, c. elegans promoters are significantly enriched in adenosine and thymidine.
these two nucleotides represent more than 66% of the total nucleotide composition in these promoter regions .
we also detected a strong t-rich region closer to the transcription start site of intestine genes, which perhaps is implicated in intestine-specific mechanisms of transcription initiation .
we then scanned these promoter regions for enriched elements uniquely present in this tissue, hoping to detect tissue-specific signatures.
we calculated the frequency of all possible hexamers within the promoter regions of our intestine and random datasets, and then gathered the frequency of these elements into six bins consisting of  <dig> nucleotides each .
among the top hits, we detected a significant enrichment of many ‘gata’ binding sites in these promoters  .
we expanded these analyses in the muscle tissues and scanned promoter regions for enriched hexamers and known trans-acting factors .
although we used unique gene datasets to search for tissue-specific motifs, the body muscle and pharynx shared several highly significant sequences, suggesting the existence of common core regulatory elements modified for pharynx and body muscle .
many eukaryotic promoters contain a ‘tataa’ binding element used to recruit the transcription machinery to the transcription start site.
when we extended this search in promoter regions in the worm genome  and in our three tissues, the frequency of the ‘tataa’ box was approximately 37%, slightly higher than what is observed in human   <cit>  , suggesting that while not ubiquitous, the tata box is still abundant in nematodes.
a list of all significantly enriched motifs detected in intestine, pharynx and body muscle promoters is shown in table s <dig> in additional file  <dig>
tissue-specific 3’end formation events are unlikely driven by unique sequence signatures in intestine and muscle tissues
we next studied changes in pas and apa in these datasets.
apa is pervasive in c. elegans  <cit> , but it is still unclear in which tissues these 3’utr isoforms are expressed, how they are produced and their consequences.
we employed an innovative library preparation method based on isothermal linear amplification of polya+ rna, which allowed us to bypass ligation-based approaches and precisely detect both the transcriptome and 3’utrome of selected tissues profiled at the same time .
this method, named spia, produces continuous linear synthesis of ssdna amplicons from a single rna template, producing consistent read numbers through the transcriptome and minimizing internal mis-priming that could generate false 3’ends during the cdna library preparation  <cit> .
using this approach, we were able to build approximately  <dig>  high-quality pas clusters  that allowed us to map 3’utr ends at single base resolution for approximately  <dig> ,  <dig> , and  <dig>  genes in our intestine and two muscle datasets, respectively .
importantly, more than 80% of these mapped 3’utr isoforms overlapped with previously described datasets  <cit>  , strongly suggesting that the vast majority of 3’utrs detected using our approach are bona fide 3’ends of mrnas.figure  <dig>
3
’utr poly-
a site mapping in tissue datasets.
we used the raw sequencing reads to map high-quality polya sites onto the ws <dig> worm annotation and compared our results with two published c. elegans 3’utrome datasets.
the number of polya clusters mapped from polya-containing sequencing reads , the portion of those that mapped to the ws <dig> worm genome annotation , the number of genes with polya sites mapped  and the number of isoforms resulting from distinct mapping of polya clusters .
the majority of mapped 3’utr isoforms are supported by two published 3’utromes and almost 90% of them are supported by at least one dataset.
left panel: the percentage of isoforms mapped to either of two published 3’utromes , to both , and those not present in either 3’utrome dataset .
right panel: the distribution of 3’utr length for all 3’utr isoforms found in each tissue dataset, along with the median  and the average length.
we then studied the length of the 3’utrs in these tissues and found that, on average, intestine genes possess shorter 3’utrs, when compared to pharynx and body muscle genes .
in mammals, shorter 3’utrs tend to escape post-transcriptional gene regulation and are more stable in comparison with longer mrnas  <cit> .
this activity has not yet been documented in worms, but presumably these short 3’utrs could lead to an increase in protein translation in the c. elegans intestine to support its diverse physiological roles.
next, we analyzed the pas elements, which are sequences in 3’utrs known to direct 3’end formation.
we superimposed our tissue-specific datasets to the worm 3’utrome from the modencode project  <cit>  and extracted the pas nucleotide composition .
the canonical pas element ‘aauaaa’ in intestine and muscle tissues was approximately 10% more abundant than in the 3’utrome overall .
the pas sequences containing one permutation of the canonical element were similar in all three tissues, while those containing two or more permutations were drastically reduced .
pas position within 3’ ends of mrnas was similar in all three tissues .
we then studied the sequence conservation near the mrna cleavage sites in genes present in each dataset, hoping to detect tissue-specific signatures .
the nucleotide frequency in these regions was remarkably similar between tissues .
we detected only a slight change in frequency of adenosines near the pas site, which was specific to 3’utrs expressed in the intestine .
taken together, our results suggest that these sequences may not contain elements important in tissue-specific 3’ end formation or that such elements are further downstream of the cleavage site and not detected by our analysis.
alternative polyadenylation is pervasive in intestine and muscle tissues
the two available worm 3’utrome datasets estimate that approximately 46% of c. elegans genes use apa  <cit> .
apa is coordinated through development, where proximal 3’utrs are expressed in earlier developmental stages and distal are expressed more frequently in later developmental stages  <cit> .
however, the extent to which apa is coordinated between c. elegans tissues and how it may participate to establish cell identity has not yet been addressed.
we employed a normalization method to select for higher confidence 3’utr isoform switching events based on the ratio between pas coverage .
intestine tissue has a larger pool of genes with two or more 3’utr isoforms , while muscle tissues mostly use single 3’utr isoforms .figure  <dig>
abundance of apa in
c
.
elegans
tissues.
a finalized list of genes with mapped 3’utr isoforms was generated for each tissue and used to compare the abundance of 3’utr isoforms between tissues.
proportion of genes subject to alternative polyadenylation in each tissue.
the intestine expressed significantly more genes containing more than one 3’utr isoform, while the muscle tissues expressed similar proportions of genes with more than one 3’utr isoform.
the average number of 3’utr isoforms detected for each gene/tissue.
the number of genes and isoforms  are displayed in each column .
we calculated and displayed the change in 3’utr isoform to gene ratio  between each tissue .
we detected slightly more apa in the intestine and pharynx, when compared with the body muscle tissue.
we reasoned that if apa is a tissue-specific event, we would be able to detect it by following the dynamics of 3’end formation in genes with one 3’utr isoform detected in each tissue.
indeed, we found that 18% to 26% of those genes switched 3’utr isoform in a tissue-specific manner ; a comprehensive list of these genes is displayed in table s <dig> in additional file  <dig>  interestingly, intestine genes more often used distal pas sites, while both muscle tissues used proximal pas sites.
when we focused this analysis comparing 3’utr switches in genes expressed in all three tissues, we found that approximately 25% of these genes use apa in a tissue-specific manner , suggesting that tissue-specific apa in worms is abundant.figure  <dig>
apa is pervasive between
c
.
elegans
tissues.
we have followed 3’utr length changes in genes with only one 3’utr isoform between intestine, pharynx and body muscle tissues.
length comparison between the same genes expressed between  intestine and pharynx,  pharynx and body muscle and  intestine and body muscle tissues.
shaded circles represent those genes expressed with proximal 3’utr isoforms in the intestine , pharynx  or body muscle , where the distal isoform was detected in the other corresponding tissue in each graph.
genes with 3’utr isoforms that were the same length between each tissue are represented in grey as noted in the legend.
distribution of unique 3’utr isoforms for genes detected in all three tissues.
the majority of these 3’utrs are common in all three tissues .
genes with a 3’utr isoform in the intestine distinct from muscle tissues are also abundant .
only 2% of these genes express different 3’utr isoforms between all three tissues .
tissue-specific 3’utr isoforms are enriched with predicted and experimentally validated mirna targets
we searched within our three datasets for commonly expressed genes with tissue-specific 3’utr isoforms .
while muscle tissues had a similar set of genes with tissue specific 3’utr isoforms, the intestine tissue had, on average, twice the amount , suggesting that the c. elegans intestine uses more apa than the two muscle tissues.figure  <dig>
analysis of tissue-specific 3’utr isoforms.
we calculated the proportions of genes in each tissue that have tissue-specific 3’utr isoforms and how many of these 3’utrs have predicted microrna targets.
charts displaying the proportion of genes containing tissue-specific 3’utr isoforms .
the intestine expresses approximately two times as many tissue-specific 3’utr isoforms as muscle tissues.
we compared the proportion of microrna targeted genes with tissue-specific 3’utrs  to the same number of randomly selected genes  in each tissue.
significantly more genes with tissue-specific 3’utr isoforms have microrna targets.
microrna targets were predicted using pictar software, using three species and five species conservation criteria, and from alg- <dig> pull-down experiments .
*p-value < <dig> , **p <  <dig> , based on two-tailed student’s t-test.
since we were able to detect widespread tissue-specific apa, we were interested to study if the genes that use tissue-specific apa were enriched with mirnas targets, and perhaps use apa to escape their regulation.
we searched in each tissue for genes with tissue-specific 3’utr isoforms that have bioinformatically predicted microrna targets .
remarkably, genes with tissue-specific 3’utr isoforms were enriched with mirna targets using both a three-species  and a five-species  conservation filter .
this is significantly more abundant than the average number of total genes expressed in each tissue having mirna targets using the same criteria .
mirna prediction software produces significantly high false and negative hits that cannot be used to properly assign targets  <cit> .
when we instead compared our dataset to in vivo mirna target footprint data from past studies  <cit> , we found a similar enrichment, with an average of 21% of genes with tissue-specific 3’utr isoforms containing validated targets .
in conclusion, these data suggest that mirna targets are much more abundant in ubiquitously expressed genes with tissue-specific 3’utr isoforms than in genes without apa, strongly linking apa to mirna targeting and post-transcriptional gene regulation.
our data support a model whereby the 3’utrs of genes with apa are regulated in a tissue-specific manner in order to evade or participate in microrna targeting.
apaome.org, a tissue transcriptome resource for c. elegans biology
we have made our data publically accessible through our apaome website  <cit> .
the apaome includes our tissue-specific datasets, as well as other important worm 3’utr datasets  <cit> , allowing the community to have a comprehensive view of apa and 3’utr biology in worms.
the apaome database  <cit>  provides detailed information on 3’utr isoforms for all protein-coding mrnas present in wormbase  <cit> , novel pictar  <cit>  and targetscan  <cit>  predictions, and includes annotations extracted from other databases as well as new annotations generated by others.
discussion
in this study, we have coupled mrna-tagging with high-throughput sequencing in a novel technique that we called pat-seq, and used it to perform an integrative analysis of the mrna transcriptome of c. elegans intestine, pharynx and body wall muscle tissues.
we have studied their transcriptome and 3’utrome at an unprecedented resolution.
in addition, since these three libraries were prepared using the same approach, we were able to directly compare the changes in gene expression and the gene content across these three somatic tissues, without extrapolating data from other studies.
our approach is an improvement over past methodologies  <cit> , allowing the identification of tissue specific mrnas at higher resolution.
pat-seq highlights c. elegans intestine and muscle transcriptome dynamics
we found that the intestine transcriptome is significantly larger than in muscle tissues, possibly to support its especially diverse physiological roles.
intestinal cells are much larger and are increasingly polyploid throughout larval development, with more transcriptional capacity compared to the smaller, diploid muscle cells  <cit> .
although there are no other comparative data in worms, recent genome-wide transcriptome analyses in the human intestine track support our findings, showing that more than 75% of all protein-coding genes are expressed in this tissue  <cit> .
the twenty large, intestinal cells may require a large pool of distinct genes to carry out functions specific to their anatomical location, since altogether these cells span from the pharynx to the posterior of the animal and the intestine is one of the largest tissues in worms.
overall, intestinal tissue is more different in gene composition than the two profiled muscle tissues.
in intestine, the most abundant genes detected are common metabolic enzymes, such as fat- <dig>  pmt- <dig>  asp- <dig>  and others, which were also detected in other available intestine-enriched datasets  <cit> .
importantly, genes and isoforms detected in our intestine dataset correlated with, and significantly expanded, previously described worm tissue-specific datasets  <cit> .
in the pharynx and body muscle, the top genes detected were myosin genes, actin isoforms and other genes.
we also detected a large number of tissue-specific alternative splicing events and fold change differences in gene expression for genes in common between tissues.
gene expression changes could be caused by stage-specific enrichments in our pull-down experiments.
we have prepared our tissue specific rnas using a well-established protocol that allows the growth of worms in liquid media  <cit> .
this protocol is known to provide an even representation of each worm developmental stage.
since all our samples were prepared using the same protocol, it is unlikely that a given sample is biased towards a specific developmental stage.
importantly, our intestine dataset overlaps consistently with recently published studies  <cit> , suggesting that if there is a general bias in all three tissue-specific datasets, it is very low.
our study detected many tissue specific genes in intestine and muscles that were previously reported by others in the same tissues .
we have also validated a selected portion of these hits using a gfp reporter approach .
we think that although very sensitive and reproducible, our pat-seq approach may introduce some noise at the lower end of detection.
further experiments may need to be performed to validate the tissue specific localization of these low expressed genes.
c. elegans promoters are at-rich and contain tissue-specific motifs
our promoter analysis showed that worm promoters are at-rich.
this result is consistent with what others have found in worm genomic regions  <cit> , drosophila  <cit> , and xenopus  <cit> , and very different from what is observed in mammalian promoters, which are gc-rich  <cit> .
gc-rich regions in promoters increase genomic thermostability  <cit> , provide more binding motifs for transcriptional activators  <cit>  and support promoter gene silencing through dna methylation at gc islands.
the at-rich nature of c. elegans promoters was previously observed in c. elegans genes expressed in the germline  <cit> , and perhaps reflects a simpler model of transcription initiation with reduced regulation.
we also demonstrate that this approach can effectively identify previously reported and novel sequence elements in tissue-specific gene datasets.
as a proof-of-concept, we detected gata transcription factors known to be critical for all aspects of c. elegans intestine development and adult function in our intestine transcriptome  <cit> , and detected potential gata sites present at a higher frequency in the promoters of intestine-specific genes.
importantly, our study highlighted the presence of many novel enriched sequence motifs, many of which have not been described yet in the literature.
while we were able to predict several transcription factors that could recognize these motifs, we still do not know if they are indeed functional, and further in vivo studies need to be performed to further characterize their role.
spia library preparation increases yield and robustness of polya sequencing
we have sequenced our tissue-specific libraries using a proprietary library preparation method, named single primer isothermal amplification , which is ideal for use with mrna-tagging, since the rna yield from this approach is typically low  <cit> .
unlike recently developed methods used to map 3’utrs, such as 3p-seq  <cit>  and fans/3’-end-seq  <cit> , spia generates cdna libraries that cover the entire transcript, allowing for more extensive downstream transcriptome analysis within the same experiment, such as coupling gene isoform mapping with the study of 3’utr dynamics.
since there is no amplification step, spia significantly minimizes internal mis-priming that could generate false 3’ends during the cdna preparation  <cit> .
it is important to note that pat-seq relies on the binding affinity of pab- <dig> to polya tails of mature mrnas, which are known to change in length in eukaryotic genes  <cit> .
this in turn can create difficulties in the quantification of gene expression levels of libraries prepared with this technology.
although this is an inherent problem in all rna-ip based approaches, our datasets correlated with previously published studies that did not use a pabpc-based approach   <cit> .
using spia, we were able to build approximately  <dig>  high-quality pas clusters that allowed us to map 3’utr ends at unprecedented resolution for approximately  <dig> ,  <dig> , and  <dig>  genes in intestine, pharynx and body muscle datasets, respectively.
importantly, we found that more than 80% of these 3’utr isoforms overlap with previously described datasets  <cit> , strongly suggesting that the vast majority of 3’utrs detected by our approach are bona fide 3’ends of mrnas.
approximately 18% to 26% of genes use tissue-specific apa
we show that approximately 18% to 26% of total genes detected in intestine and muscle tissues used tissue-specific apa.
intestine genes seemed to favor proximal-to-distal pas switches, leading to longer 3’utr isoforms, while both muscle tissues alternated 3’utr isoforms at a similar rate.
previous studies of 3’utr datasets reported that as many as 46% of worm genes use apa across multiple tissues and developmental stages  <cit> .
this apparent discordance with our findings indicates that a large majority of genes in worms use only one 3’utr isoform in a given tissue, suggesting that apa is indeed an important mechanism used by cells to regulate gene expression at the tissue-specific level.
importantly, our work identified an overall high number of novel 3’utr isoforms that were not present in past analyses  <cit> .
this pool spans from 9% to 16%, depending of the tissue examined .
previous work reported that the worm 3’utrome is not saturated and other novel 3’utr isoforms may be present  <cit> .
interestingly, the majority of the 3’utr isoforms within this pool are tissue-specific , suggesting that perhaps these 3’utr isoforms are also rare and were not identified in earlier studies because of the limit of sensitivity of their mixed-tissue, transcriptome-wide approaches  <cit> .
our analysis uncovered significant apa in worm tissues, but we could not identify upstream tissue-specific elements involved in 3’end formation, suggesting that in worms, other accessory tissue-specific factors  <cit>  or their dosage  <cit>  may play a role instead  <cit> .
tissue-specific 3’utr isoforms are linked to microrna regulation
past dogma that the protein and the transcription levels in cells are directly proportional is not accurate anymore  <cit> .
thanks to the introduction of novel high-throughput technologies, it is now clear that there is not a direct correlation between the transcriptomes and the proteomes of cells or tissues.
instead, mirnas, together with other ncrnas and rna binding proteins, play key roles in modulating the final gene output on its way to protein expression  <cit> .
this modulation, when combined with the abundance of apa detected in this study, suggests a more complex picture, where there are not only negative regulatory networks through mirnas, but also novel unexplored positive regulatory networks operated though apa.
these positive networks are driven by genes that switch between 3’utr isoforms to escape mirna targeting, allowing their expression.
in this view, both mirnas and apa can, in principle, dramatically reshape gene expression output, implying they both play key roles in the establishment and maintenance of cell and tissue identity.
in this study we found that genes with tissue-specific 3’utr isoforms are enriched in microrna targets using both a three-species  and a five-species  conservation criteria.
this was significantly more abundant than what we saw in randomly selected 3’utr isoforms using a three-species  and a five-species  conservation criteria in each tissue.
we also found a similar enrichment comparing our dataset to the experimentally validated alg- <dig> footprints  <cit> .
our results in three worm somatic tissues link mirna regulation to apa, showing that microrna targets are much more abundant in ubiquitously expressed genes with tissue-specific 3’utr isoforms than in genes that do not use apa.
recently, microrna populations from intestine and body muscle tissues were isolated using an rna-ip strategy, providing a tissue-level atlas of microrna expression  <cit> .
unfortunately, while this study suggests that mirnas are also pervasive in worm tissues, it is still unclear which genes they target, and further experiments have to be performed to highlight these regulatory networks.
apaome.org: a resource for 3’utr biology
we have compiled our tissue-specific transcriptomes into a useful online resource for scientists interested in 3’utr biology and apa.
the apaome.org site uses an apache web server and several custom-made perl scripts that query a dedicated mysql database.
it is currently hosted in the biodesign institute at arizona state university, and offers a simple and well-integrated interactive user interface to query gene records and 3’utr isoform data, giving access to a dedicated gbrowse installation specifically designed to study apa in worms.
this database displays tracks for each tissue transcriptome, including tissue-specific apa, as well as curated 3’utr data from previously published studies  <cit> .
CONCLUSIONS
here we present the first comparative analysis of the transcriptome, the 3’utrome, and the promoter diversity of three large somatic tissues of the soil nematode c. elegans.
we found abundant tissue-specific gene expression changes that correlated with the presence of distinct promoter signatures between c. elegans intestine and muscle tissues, and defined thousands of tissue-specific splice isoforms.
we have discovered that tissue-specific apa is pervasive in nematodes, and that 3’utr isoform changes correlated with gain or loss of mirna target elements, suggesting a role for apa in modulating tissue-specific post-transcriptional gene regulation.

 transcriptome plasticity is a powerful modulator of gene expression that provides multicellular organisms with the complexity needed to drive development and maintain tissue identity. apart from a few cases, we still do not fully understand what specific transcriptome rearrangements occur in somatic tissues, and how they integrate with gene regulation at the post-transcriptional level to maintain tissue and cellular diversity. the small nematode caenorhabditis elegans is an ideal model organism to study these events, since its gene model has been extensively characterized in past years. it is also experimentally tractable with short and precise developmental timing, approximately somatic cells, a transparent and simple body plan and an entirely defined cell lineage. large-scale efforts have detailed its transcriptome at a global level. promoter diversity, alternate splicing events and changes in 3’ untranslated regions are also well characterized. while the formation of several worm tissues and the genes involved in driving these processes have been extensively described, we still do not fully understand how the synergistic activity of tissue-specific events before, during and after transcription drive and maintain tissue identity. pre-transcriptionally, enrichment of sequence-specific elements within c. elegans promoters has been linked to tissue-specific changes in gene expression, suggesting that these elements, together with trans-acting factors that recognize them, are fundamental for driving the transcriptional programs of unique tissues. during transcription, mrna isoforms resulting from alternative splicing increase transcriptome complexity, coordinating tissue development. recently, a genome-wide study in c. elegans found that thousands of transcripts are alternatively spliced and many of them change splicing patterns during development, suggesting that tissue-specific splicing may play key roles in this process. post-transcriptionally, 3’utrs are known to contain multiple regulatory sequence elements important for gene regulation. recently, two independent studies suggest that more than 40% of worm genes possess 3’utrs subjected to alternative polyadenylation, a mechanism that generates multiple 3’utr isoforms for the same genes. this process is widespread in metazoans, coordinated through development, and misregulated in disease, underscoring a potential role for apa in tissue-specific modulation of gene expression. the cleavage and polyadenylation of nascent mrnas in eukaryotes is mainly executed by two large multimeric complexes named cleavage and polyadenylation specificity factor and cleavage stimulation factor. cpsf recognizes and binds to the polyadenylation ) signal element located approximately nt from the polya site in the 3’utr of mrnas. in metazoans, the pas sequence is commonly aauaaa. this sequence is necessary and sufficient for 3’end polyadenylation. cstf directly interacts with cpsf and binds to gu-rich elements downstream of the cleavage site. although apa is pervasive in worms and correlated with development, suggesting that apa functions in worms tissues, it is unclear whether apa is tissue-specific. both cpsf and cstf are likely to have a role in managing the choice between pas elements in the same 3’utr and inducing apa. there may also be additional tissue-specific accessory factors that modify the basal polyadenylation machinery, controlling the usage of one pas element over another. tissue-specific isoforms of the cpsf or cstf complexes could be responsible for apa. over a decade ago, stoichiometric levels of cstf members were indeed shown to control apa in b cell activation, and recent high-throughput approaches showed that other factors might also play important roles in modulating apa. other processing factors were also recently shown to influence the location of cleavage. these studies underscore the importance of the correct stoichiometric ratio of each of the 3’end processing factors for producing a mature mrna. surprisingly, it was also recently shown that u snrnp is involved in this process, suggesting possible cross talk between apa and the rna splicing machinery. these models may not be mutually exclusive. in c. elegans, the isolation of tissue-specific mrna to study transcriptome plasticity and apa is challenging due to the lack of in vitro cell cultures, the worm’s tough outer cuticle that interferes with sample preparation and the small size of many tissues that prevents manual dissection. several techniques have been developed to circumvent these issues, including fluorescence-activated cell and nuclear sorting, nuclei-tagging and mrna-tagging. in particular, mrna-tagging has been widely used to isolate and study mrna from muscle, epithelial, hypodermal, neuronal and seam cells. this technique uses tissue-specific promoters to drive expression of a flag epitope-tagged cytoplasmic poly-a binding protein, which specifically binds to the poly-tail of mrnas in the cytoplasm, followed by crosslinking and immunoprecipitation of tissue-specific mrnas. thus far, mrna-tagging has been coupled with dna microarrays and genomic tiling arrays, which both lack the advanced sensitivity and specificity possible today with deep sequencing, potentially limiting the results obtained with this methodology. recently, a novel method that couples tissue-specific nuclei isolation with deep sequencing was used to analyze the c. elegans intestine transcriptome. unfortunately, while enhancing the sensitivity of tissue-specific mrna extraction and sequencing, this technique limits the analysis to nuclear mrna species, and only identifies short 3’end portions of 3’utrs that need to be bioinformatically attached to their closest genes, potentially introducing mapping inaccuracies. in addition, this method does not provide tissue-specific mrna isoform data, losing an important component needed to study the transcriptome plasticity of individual tissues. integrating mrna-tagging with rna-seq analysis could significantly improve the resolution of these studies and identify additional factors controlling tissue development and identity. here, we have improved tissue-specific transcriptome profiling in c. elegans, optimizing mrna-tagging for deep sequencing. we call this updated method polya-tagging and sequencing. we have applied pat-seq and profiled tissue-specific mrna from the c. elegans intestine and two muscle tissues belonging to the pharynx and body wall of mixed stage worms. we describe and compare gene expression, promoter sequence composition, mrna isoforms changes and alternative polyadenylation between each tissue. pat-seq significantly improved the resolution of tissue-specific transcriptomes from previous studies, adding thousands of novel genes and isoforms that allow for a more comprehensive analysis of them. in addition, we have used pat-seq to profile the transcriptome of a previously uncharacterized tissue, allowing us for the first time to directly compare gene expression changes between different tissues from the same organism at a higher resolution, using the same experimental settings. we find that transcript diversity detected among these three tissues is mirrored by the presence of characteristic tissue-specific promoter signatures. in addition, we found that apa is widely used at a tissue-specific level, highlighting major complex tissue-specific transcript dynamics and post-transcriptional regulatory mechanisms. we describe a large number of 3’utr isoforms specifically expressed in each tissue and find that these 3’utrs are enriched for experimentally and bioinformatically predicted microrna targets, suggesting that tissue-specific apa is used in worms as a mechanism to interface with mirna mediated post-transcriptional gene regulation. finally, we have remapped, incorporated and curated 3’utr data from previously published studies and integrated these data with our new tissue-specific datasets. the database is accessible through our new worm apa-specific website, which is publically available and represents a unique resource for the scientists interested in 3’utr biology.  isolation of mrnas from intestine and muscle tissues mrna-tagging has so far been coupled with low-resolution platforms, such as microarrays and tiling arrays, that lack the sensitivity required to detect low expressed transcripts, pinpoint gene isoform changes and map 3’utrs at single base resolution. to improve upon its sensitivity and specificity, we made several key changes to the original mrna-tagging protocol: 1) we added three tandem flag-epitope tags instead of one, to improve the efficiency of the flag pull-down. 2) in the original mrna-tagging protocol, the flag-tagged pabpc construct is selectively expressed as extra chromosomal arrays, which are unstable and often lead to mosaic expression patterns, or integrated as multiple copy lines. we instead opted for the widely used mos- single copy insertion technology, which stably incorporates the construct of interest in the worm genome. 3) we adopted a novel strategy to prepare the tissue-specific cdna libraries that relies on linear amplification of mrnas, minimizing the quantification error rate due to limited starting material, providing high-quality transcriptome and 3’end data in the same experiment; and 4) replaced the microarray step with next generation sequencing to improve data resolution. we used tissue-specific promoters to drive the expression of the c. elegans cytoplasmic pabpc gene, in-frame with gfp and fused to a 3 × flag tag, in intestine, pharynx or body muscle. as mossci technology mediates transgene and rescue cassette insertion into a specified region of chromosome ii, we confirmed this integration event in each transgenic worm line and verified its expression using western blotting.figure pat-seq of intestine and muscles tissues. pat-seq uses gateway-compatible entry vectors expressing the polya-pull cassette in each tissue using tissue-specific promoters. polya-pull expressed in the intestine, pharynx, and body muscle. expression of polya-pull produces a 3 × flag-tag fused to pab-, which specifically binds to the poly tails of mrnas. the complex is immunoprecipitated using α-flag beads. tissue-specific cdna libraries are sequenced and mapped onto the ws gene model. detection of stable integration of the polya-pull cassette. left panel: using pcr we detected genomic integration of the common portion of the polya-pull cassette in each tissue. the negative control, myo-2Δpab- was also integrated. right panel: western blotting using α-flag antibodies detected the in-frame polya-pull fusion protein in lysates from transgenic worms expressing it in the pharynx but not in lysate from wild type n worms. quantification of the specificity and sensitivity of the pull-down using rt-pcr: left panel: myo-, ges- and dpy- transcripts were detected in total rna extracted from wild type n worms. middle panel: using immunoprecipitation, we successfully detected the presence of the muscle-specific gene myo- and the exogenous unc- 3’utr, but not the intestine-specific ges- and the hypodermis-specific dpy-. these transgenic worms expressed polya-pull cassette in the pharynx, but not in our negative control in wild type n worms. the primers used to detect unc- 3’utr also detected 18s rrnas. this band was replaced with two unc- 3’utr isoforms, suggesting that polya-pull enriched for polya+ rnas. right panel: we are unable to isolate tissue-specific rna from worms lacking pab-. we tested the sensitivity and tissue-specificity of our mrna pull-down approach using worms expressing polya-pull in the pharynx, validating the ability of our construct to selectively bind muscle specific transcripts. the known intestine-specific transcript ges- and hypodermis-specific dpy- were depleted from the same sample. immunoprecipitation from wild type n worms yielded no detectable background in rt-pcr for the same genes. to test if our polya-pull construct selectively binds polya+ rnas, we prepared a gfp::3 × flag fusion protein that does not contain pab- this vector is unable to bind polyadenylated mrnas. we expressed this new construct in the pharynx. as expected, using this construct, we were unable to detect the pharynx-specific myo- transcript. taken together, these results suggest that our polya-pull construct effectively enriches tissue-specific mrnas and specifically binds polya+ rnas. pat-seq analysis of mrnas from intestine and muscle tissues we then prepared our tissue-specific libraries with two biological replicates. we also performed two negative control pull-down experiments expressing the Δpab-1-pull construct in the pharynx to optimize the sensitivity and specificity of our approach. following the pull-down, the cdna libraries were prepared using isothermal linear cdna amplifications, which allows cdna synthesis across the full length of the transcripts with as little as ng of total rna, thus improving the coverage of our whole-transcriptome amplification independently of the 3’polya tail. we, then, barcoded, pooled and sequenced our eight rna pull-down libraries on the illumina hiseq- platform. the resulting paired reads were computationally assembled and mapped onto the c. elegans ws genome. a summary of the results of this mapping is displayed in table s in additional file we obtained approximately m unique reads per sample. the number of genes mapped in each tissue was consistent between biological replicates, reflecting the robustness of our library preparation from tissue-specific rna samples. the overall number of genes and their ratio detected in each biological replicate, with the exception of our negative control, was comparable, suggesting that our approach was consistent. in the intestine, we detected a much larger number of genes compared with that of pharynx and body muscle tissues. genes and their expression levels between biological replicates correlated well, with the exception of the myo2p::Δpab- control, further supporting the reproducibility of our approach and suggesting that pat-seq is specific and sensitive. a closer look into transcripts recovered with myo2p::Δpab- revealed an enrichment of ncrnas and other common contaminants, reinforcing that this sample represented random non-specific rna pulled-down during the immunoprecipitation step. we have validated the tissue localization of selected tissue-specific genes identified with pat-seq by cloning their promoters and using them to drive expression of gfp in vivo. we detected a common core set of approximately unique genes present in all three tissues. these transcripts include housekeeping genes, such as actin, histone genes, ribosomal proteins, genes involved in transcription, basal transcription factors, and genes involved in dna maintenance and replication. a complete list of genes and isoforms detected in each tissue is shown as a diagram in figure  top panel and comprehensively in table s in additional file figure distribution of tissue -specific gene expression and alternative polyadenylation in intestine, pharynx and body muscle. top panel: tissue-specific genes identified by pat-seq and the distribution of their expression levels between each tissue. a large pool of genes is uniquely expressed in the intestine, while a smaller portion of and genes is expressed uniquely in the pharynx and body muscle, respectively. we have detected a common set of genes expressed in all three tissues. edges represent the presence of transcripts in each tissue, and color-coding indicates expression levels of genes in tissues. bottom panel: the genes shared in all three tissues were further sorted based on the 3’utr isoform and their expression levels. approximately 25% to 30% of these genes use common 3’ends in these three tissues, while the remaining 70% use tissue-specific 3’utr isoforms. the intestine transcriptome is expansive, expressing over 30% of c. elegans mrnas the intestine is one of the largest tissues in c. elegans, composed of large cells and a total of to nuclei, with a final 32-fold polyploidy in the adult worm. it is also one of the most functionally diverse tissues, participating in digestion, nutrient transport and storage, innate immunity, response to environmental toxins, defecation and dauer formation. while the intestine transcriptome has been studied extensively, our results correlated with and significantly expanded these studies. we detected a total of expressed genes in this tissue, of which genes and spliced isoforms are uniquely expressed in the intestine, but not in either muscle tissue. the most abundant in this dataset were metabolic enzymes and nutrient transport genes, consistent with this tissue’s physiological function in digestion. among these intestine-only genes, we identified unique transcripts that contain a dna binding domain and were previously described as transcription factors. we speculate that these transcription factors may contribute to the gene regulatory network necessary for tissue identity and function. as expected, members of the gata family are among the most abundant transcription factors. these factors bind ‘gata’ elements on promoters and have been shown to regulate endoderm specification and all aspects of intestine development and function in c. elegans. in addition, a significant portion of all transcription factors uniquely expressed in this tissue are members of the nuclear hormone receptor family. many members of this class of transcription factors were also previously shown to regulate c. elegans metabolism. we also detected a large pool of novel intestine-specific transcription factors with unknown roles that need to be further investigated. recently, pauli and colleagues coupled mrna-tagging with dna microarrays from l4-stage worms and identified intestine transcripts. others produced a sage library of transcripts from dissected worm intestines from mutant adults, and detected a total of intestine genes. a comparison of each of these datasets with ours identified 71% and 78% overlap with mcghee and pauli datasets, respectively, suggesting that the increased sensitivity of pat-seq applied to study the intestine transcriptome of mixed stage worms expanded the core c. elegans intestine transcriptome. haenni and colleagues recently optimized a procedure for extracting intact nuclei from c. elegans intestine, followed by fluorescence-activated cell sorting and deep sequencing of mrna, focusing on the 3’ end of the transcriptome. this approach allowed the authors to map genes expressed in this tissue. we compared our intestine datasets with these results, downloading and remapping the raw reads from haenni et al. using the same filtering criteria used in our datasets. we found that 71% of genes were detected in both datasets, leaving 29% of genes present in the haenni et al. dataset not present in ours. despite these differences, the distribution of genes detected in both datasets plotted by expression values correlated. our inability to detect 1/ of the genes in haenni et al. may be attributed to the fundamentally different techniques used in the preparation of our cytoplasmic, intestine-specific mrnas using pat-seq versus nuclear cdna prepared from isolated facs sorted nuclei as in haenni et al.. the difference in gene pools may have arisen because of the different cellular origin of the mrnas analyzed by the two studies. finally, we have overlapped our datasets with mcghee et al. and haenni et al., the two intestine transcriptomes datasets obtained by sequencing, revealing a core set of shared genes. these genes represent a collection of high confidence intestine expressed genes, supported by at least three independent approaches, and will provide important insights in unraveling the genetic basis of intestine tissue identity in worms. muscle transcriptomes are smaller but contain mostly unique gene pools c. elegans possess only two large muscles: the pharynx and the body muscle. the pharynx, or foregut, is an important developmental model composed of eight layers of muscle, in addition to surrounding neural and epithelial tissue. the muscle component of this tissue is composed of cells that coordinate intake and physical crushing of the worm bacterial diet, subsequently facilitating raw nutrient transfer to the intestinal lumen for digestion. while the genetic factors controlling early development of the pharynx have been described in detail, individual cell-types of the pharynx are less characterized because genetic signatures belonging to these specific subgroups have not been extensively studied. we detected genes expressed in pharynx muscle. among the top genes expressed were several myosin and actin isoforms, and pharynx-specific neurotransmitters, consistent with this tissue’s muscular identity. importantly, we found only unique genes with spliced isoforms significantly expressed in this tissue. most of these genes have unknown function, with only transcripts described in wormbase so far. the top genes of this list are collagen isoforms and motor protein genes. within this pool we also detected pharynx-specific transcription factors. their gene targets are mostly unknown. the body muscle tissue, defined as all non-pharyngeal muscle cells, is homologous to vertebrate skeletal muscle, and critical for locomotion, egg laying, defecation, and mating. several groups also studied the transcriptome of this tissue. we detected genes expressed in the body muscle. similar to our pharynx dataset, within this pool we detected only unique genes corresponding to spliced isoforms. the list of top ten genes in the body wall muscle dataset was also enriched for muscle-specific genes such as myosin and actin isoforms. we also detected a unique gene pool, including previously identified genes, such as those in the calveolin family, and type iv collagen. out of predicted worm transcription factors, we identified genes that are uniquely expressed in this tissue, including unc- a srf-like transcription factor essential for body wall muscle development, and blmp- a prdm family member required for embryonic slow muscle fiber formation in vertebrates. using pat-seq we were able to detect many statistically significant isoform expression changes between intestine and muscle tissues. tissue-specific promoters possess unique signatures the compact nature of the promoter regions in the c. elegans genome provides us with a unique platform to examine tissue-specific elements in these regions and highlight signatures such as transcription factor binding sites. we first studied the sequence composition of these promoters, defined as the portion of genomic sequences from - to + from the tss of protein coding genes. we compared these sequences to promoters of random genes from the whole c. elegans transcriptome. we found that, contrary to higher metazoans, such as in humans where promoters are significantly enriched in cytosines and guanosines, c. elegans promoters are significantly enriched in adenosine and thymidine. these two nucleotides represent more than 66% of the total nucleotide composition in these promoter regions. we also detected a strong t-rich region closer to the transcription start site of intestine genes, which perhaps is implicated in intestine-specific mechanisms of transcription initiation. we then scanned these promoter regions for enriched elements uniquely present in this tissue, hoping to detect tissue-specific signatures. we calculated the frequency of all possible hexamers within the promoter regions of our intestine and random datasets, and then gathered the frequency of these elements into six bins consisting of nucleotides each. among the top hits, we detected a significant enrichment of many ‘gata’ binding sites in these promoters. we expanded these analyses in the muscle tissues and scanned promoter regions for enriched hexamers and known trans-acting factors. although we used unique gene datasets to search for tissue-specific motifs, the body muscle and pharynx shared several highly significant sequences, suggesting the existence of common core regulatory elements modified for pharynx and body muscle. many eukaryotic promoters contain a ‘tataa’ binding element used to recruit the transcription machinery to the transcription start site. when we extended this search in promoter regions in the worm genome and in our three tissues, the frequency of the ‘tataa’ box was approximately 37%, slightly higher than what is observed in human, suggesting that while not ubiquitous, the tata box is still abundant in nematodes. a list of all significantly enriched motifs detected in intestine, pharynx and body muscle promoters is shown in table s in additional file tissue-specific 3’end formation events are unlikely driven by unique sequence signatures in intestine and muscle tissues we next studied changes in pas and apa in these datasets. apa is pervasive in c. elegans, but it is still unclear in which tissues these 3’utr isoforms are expressed, how they are produced and their consequences. we employed an innovative library preparation method based on isothermal linear amplification of polya+ rna, which allowed us to bypass ligation-based approaches and precisely detect both the transcriptome and 3’utrome of selected tissues profiled at the same time. this method, named spia, produces continuous linear synthesis of ssdna amplicons from a single rna template, producing consistent read numbers through the transcriptome and minimizing internal mis-priming that could generate false 3’ends during the cdna library preparation. using this approach, we were able to build approximately high-quality pas clusters that allowed us to map 3’utr ends at single base resolution for approximately,, and genes in our intestine and two muscle datasets, respectively. importantly, more than 80% of these mapped 3’utr isoforms overlapped with previously described datasets, strongly suggesting that the vast majority of 3’utrs detected using our approach are bona fide 3’ends of mrnas.figure 3 ’utr poly- a site mapping in tissue datasets. we used the raw sequencing reads to map high-quality polya sites onto the ws worm annotation and compared our results with two published c. elegans 3’utrome datasets. the number of polya clusters mapped from polya-containing sequencing reads, the portion of those that mapped to the ws worm genome annotation, the number of genes with polya sites mapped and the number of isoforms resulting from distinct mapping of polya clusters. the majority of mapped 3’utr isoforms are supported by two published 3’utromes and almost 90% of them are supported by at least one dataset. left panel: the percentage of isoforms mapped to either of two published 3’utromes, to both, and those not present in either 3’utrome dataset. right panel: the distribution of 3’utr length for all 3’utr isoforms found in each tissue dataset, along with the median and the average length. we then studied the length of the 3’utrs in these tissues and found that, on average, intestine genes possess shorter 3’utrs, when compared to pharynx and body muscle genes. in mammals, shorter 3’utrs tend to escape post-transcriptional gene regulation and are more stable in comparison with longer mrnas. this activity has not yet been documented in worms, but presumably these short 3’utrs could lead to an increase in protein translation in the c. elegans intestine to support its diverse physiological roles. next, we analyzed the pas elements, which are sequences in 3’utrs known to direct 3’end formation. we superimposed our tissue-specific datasets to the worm 3’utrome from the modencode project and extracted the pas nucleotide composition. the canonical pas element ‘aauaaa’ in intestine and muscle tissues was approximately 10% more abundant than in the 3’utrome overall. the pas sequences containing one permutation of the canonical element were similar in all three tissues, while those containing two or more permutations were drastically reduced. pas position within 3’ ends of mrnas was similar in all three tissues. we then studied the sequence conservation near the mrna cleavage sites in genes present in each dataset, hoping to detect tissue-specific signatures. the nucleotide frequency in these regions was remarkably similar between tissues. we detected only a slight change in frequency of adenosines near the pas site, which was specific to 3’utrs expressed in the intestine. taken together, our results suggest that these sequences may not contain elements important in tissue-specific 3’ end formation or that such elements are further downstream of the cleavage site and not detected by our analysis. alternative polyadenylation is pervasive in intestine and muscle tissues the two available worm 3’utrome datasets estimate that approximately 46% of c. elegans genes use apa. apa is coordinated through development, where proximal 3’utrs are expressed in earlier developmental stages and distal are expressed more frequently in later developmental stages. however, the extent to which apa is coordinated between c. elegans tissues and how it may participate to establish cell identity has not yet been addressed. we employed a normalization method to select for higher confidence 3’utr isoform switching events based on the ratio between pas coverage. intestine tissue has a larger pool of genes with two or more 3’utr isoforms, while muscle tissues mostly use single 3’utr isoforms.figure abundance of apa in c. elegans tissues. a finalized list of genes with mapped 3’utr isoforms was generated for each tissue and used to compare the abundance of 3’utr isoforms between tissues. proportion of genes subject to alternative polyadenylation in each tissue. the intestine expressed significantly more genes containing more than one 3’utr isoform, while the muscle tissues expressed similar proportions of genes with more than one 3’utr isoform. the average number of 3’utr isoforms detected for each gene/tissue. the number of genes and isoforms are displayed in each column. we calculated and displayed the change in 3’utr isoform to gene ratio between each tissue. we detected slightly more apa in the intestine and pharynx, when compared with the body muscle tissue. we reasoned that if apa is a tissue-specific event, we would be able to detect it by following the dynamics of 3’end formation in genes with one 3’utr isoform detected in each tissue. indeed, we found that 18% to 26% of those genes switched 3’utr isoform in a tissue-specific manner; a comprehensive list of these genes is displayed in table s in additional file interestingly, intestine genes more often used distal pas sites, while both muscle tissues used proximal pas sites. when we focused this analysis comparing 3’utr switches in genes expressed in all three tissues, we found that approximately 25% of these genes use apa in a tissue-specific manner, suggesting that tissue-specific apa in worms is abundant.figure apa is pervasive between c. elegans tissues. we have followed 3’utr length changes in genes with only one 3’utr isoform between intestine, pharynx and body muscle tissues. length comparison between the same genes expressed between intestine and pharynx, pharynx and body muscle and intestine and body muscle tissues. shaded circles represent those genes expressed with proximal 3’utr isoforms in the intestine, pharynx or body muscle, where the distal isoform was detected in the other corresponding tissue in each graph. genes with 3’utr isoforms that were the same length between each tissue are represented in grey as noted in the legend. distribution of unique 3’utr isoforms for genes detected in all three tissues. the majority of these 3’utrs are common in all three tissues. genes with a 3’utr isoform in the intestine distinct from muscle tissues are also abundant. only 2% of these genes express different 3’utr isoforms between all three tissues. tissue-specific 3’utr isoforms are enriched with predicted and experimentally validated mirna targets we searched within our three datasets for commonly expressed genes with tissue-specific 3’utr isoforms. while muscle tissues had a similar set of genes with tissue specific 3’utr isoforms, the intestine tissue had, on average, twice the amount, suggesting that the c. elegans intestine uses more apa than the two muscle tissues.figure analysis of tissue-specific 3’utr isoforms. we calculated the proportions of genes in each tissue that have tissue-specific 3’utr isoforms and how many of these 3’utrs have predicted microrna targets. charts displaying the proportion of genes containing tissue-specific 3’utr isoforms. the intestine expresses approximately two times as many tissue-specific 3’utr isoforms as muscle tissues. we compared the proportion of microrna targeted genes with tissue-specific 3’utrs to the same number of randomly selected genes in each tissue. significantly more genes with tissue-specific 3’utr isoforms have microrna targets. microrna targets were predicted using pictar software, using three species and five species conservation criteria, and from alg- pull-down experiments. *p-value <, **p < , based on two-tailed student’s t-test. since we were able to detect widespread tissue-specific apa, we were interested to study if the genes that use tissue-specific apa were enriched with mirnas targets, and perhaps use apa to escape their regulation. we searched in each tissue for genes with tissue-specific 3’utr isoforms that have bioinformatically predicted microrna targets. remarkably, genes with tissue-specific 3’utr isoforms were enriched with mirna targets using both a three-species and a five-species conservation filter. this is significantly more abundant than the average number of total genes expressed in each tissue having mirna targets using the same criteria. mirna prediction software produces significantly high false and negative hits that cannot be used to properly assign targets. when we instead compared our dataset to in vivo mirna target footprint data from past studies, we found a similar enrichment, with an average of 21% of genes with tissue-specific 3’utr isoforms containing validated targets. in conclusion, these data suggest that mirna targets are much more abundant in ubiquitously expressed genes with tissue-specific 3’utr isoforms than in genes without apa, strongly linking apa to mirna targeting and post-transcriptional gene regulation. our data support a model whereby the 3’utrs of genes with apa are regulated in a tissue-specific manner in order to evade or participate in microrna targeting. apaome.org, a tissue transcriptome resource for c. elegans biology we have made our data publically accessible through our apaome website. the apaome includes our tissue-specific datasets, as well as other important worm 3’utr datasets, allowing the community to have a comprehensive view of apa and 3’utr biology in worms. the apaome database provides detailed information on 3’utr isoforms for all protein-coding mrnas present in wormbase, novel pictar and targetscan predictions, and includes annotations extracted from other databases as well as new annotations generated by others. discussion in this study, we have coupled mrna-tagging with high-throughput sequencing in a novel technique that we called pat-seq, and used it to perform an integrative analysis of the mrna transcriptome of c. elegans intestine, pharynx and body wall muscle tissues. we have studied their transcriptome and 3’utrome at an unprecedented resolution. in addition, since these three libraries were prepared using the same approach, we were able to directly compare the changes in gene expression and the gene content across these three somatic tissues, without extrapolating data from other studies. our approach is an improvement over past methodologies, allowing the identification of tissue specific mrnas at higher resolution. pat-seq highlights c. elegans intestine and muscle transcriptome dynamics we found that the intestine transcriptome is significantly larger than in muscle tissues, possibly to support its especially diverse physiological roles. intestinal cells are much larger and are increasingly polyploid throughout larval development, with more transcriptional capacity compared to the smaller, diploid muscle cells. although there are no other comparative data in worms, recent genome-wide transcriptome analyses in the human intestine track support our findings, showing that more than 75% of all protein-coding genes are expressed in this tissue. the twenty large, intestinal cells may require a large pool of distinct genes to carry out functions specific to their anatomical location, since altogether these cells span from the pharynx to the posterior of the animal and the intestine is one of the largest tissues in worms. overall, intestinal tissue is more different in gene composition than the two profiled muscle tissues. in intestine, the most abundant genes detected are common metabolic enzymes, such as fat- pmt- asp- and others, which were also detected in other available intestine-enriched datasets. importantly, genes and isoforms detected in our intestine dataset correlated with, and significantly expanded, previously described worm tissue-specific datasets. in the pharynx and body muscle, the top genes detected were myosin genes, actin isoforms and other genes. we also detected a large number of tissue-specific alternative splicing events and fold change differences in gene expression for genes in common between tissues. gene expression changes could be caused by stage-specific enrichments in our pull-down experiments. we have prepared our tissue specific rnas using a well-established protocol that allows the growth of worms in liquid media. this protocol is known to provide an even representation of each worm developmental stage. since all our samples were prepared using the same protocol, it is unlikely that a given sample is biased towards a specific developmental stage. importantly, our intestine dataset overlaps consistently with recently published studies, suggesting that if there is a general bias in all three tissue-specific datasets, it is very low. our study detected many tissue specific genes in intestine and muscles that were previously reported by others in the same tissues. we have also validated a selected portion of these hits using a gfp reporter approach. we think that although very sensitive and reproducible, our pat-seq approach may introduce some noise at the lower end of detection. further experiments may need to be performed to validate the tissue specific localization of these low expressed genes. c. elegans promoters are at-rich and contain tissue-specific motifs our promoter analysis showed that worm promoters are at-rich. this result is consistent with what others have found in worm genomic regions, drosophila, and xenopus, and very different from what is observed in mammalian promoters, which are gc-rich. gc-rich regions in promoters increase genomic thermostability, provide more binding motifs for transcriptional activators and support promoter gene silencing through dna methylation at gc islands. the at-rich nature of c. elegans promoters was previously observed in c. elegans genes expressed in the germline, and perhaps reflects a simpler model of transcription initiation with reduced regulation. we also demonstrate that this approach can effectively identify previously reported and novel sequence elements in tissue-specific gene datasets. as a proof-of-concept, we detected gata transcription factors known to be critical for all aspects of c. elegans intestine development and adult function in our intestine transcriptome, and detected potential gata sites present at a higher frequency in the promoters of intestine-specific genes. importantly, our study highlighted the presence of many novel enriched sequence motifs, many of which have not been described yet in the literature. while we were able to predict several transcription factors that could recognize these motifs, we still do not know if they are indeed functional, and further in vivo studies need to be performed to further characterize their role. spia library preparation increases yield and robustness of polya sequencing we have sequenced our tissue-specific libraries using a proprietary library preparation method, named single primer isothermal amplification, which is ideal for use with mrna-tagging, since the rna yield from this approach is typically low. unlike recently developed methods used to map 3’utrs, such as 3p-seq and fans/3’-end-seq, spia generates cdna libraries that cover the entire transcript, allowing for more extensive downstream transcriptome analysis within the same experiment, such as coupling gene isoform mapping with the study of 3’utr dynamics. since there is no amplification step, spia significantly minimizes internal mis-priming that could generate false 3’ends during the cdna preparation. it is important to note that pat-seq relies on the binding affinity of pab- to polya tails of mature mrnas, which are known to change in length in eukaryotic genes. this in turn can create difficulties in the quantification of gene expression levels of libraries prepared with this technology. although this is an inherent problem in all rna-ip based approaches, our datasets correlated with previously published studies that did not use a pabpc-based approach. using spia, we were able to build approximately high-quality pas clusters that allowed us to map 3’utr ends at unprecedented resolution for approximately,, and genes in intestine, pharynx and body muscle datasets, respectively. importantly, we found that more than 80% of these 3’utr isoforms overlap with previously described datasets, strongly suggesting that the vast majority of 3’utrs detected by our approach are bona fide 3’ends of mrnas. approximately 18% to 26% of genes use tissue-specific apa we show that approximately 18% to 26% of total genes detected in intestine and muscle tissues used tissue-specific apa. intestine genes seemed to favor proximal-to-distal pas switches, leading to longer 3’utr isoforms, while both muscle tissues alternated 3’utr isoforms at a similar rate. previous studies of 3’utr datasets reported that as many as 46% of worm genes use apa across multiple tissues and developmental stages. this apparent discordance with our findings indicates that a large majority of genes in worms use only one 3’utr isoform in a given tissue, suggesting that apa is indeed an important mechanism used by cells to regulate gene expression at the tissue-specific level. importantly, our work identified an overall high number of novel 3’utr isoforms that were not present in past analyses. this pool spans from 9% to 16%, depending of the tissue examined. previous work reported that the worm 3’utrome is not saturated and other novel 3’utr isoforms may be present. interestingly, the majority of the 3’utr isoforms within this pool are tissue-specific, suggesting that perhaps these 3’utr isoforms are also rare and were not identified in earlier studies because of the limit of sensitivity of their mixed-tissue, transcriptome-wide approaches. our analysis uncovered significant apa in worm tissues, but we could not identify upstream tissue-specific elements involved in 3’end formation, suggesting that in worms, other accessory tissue-specific factors or their dosage may play a role instead. tissue-specific 3’utr isoforms are linked to microrna regulation past dogma that the protein and the transcription levels in cells are directly proportional is not accurate anymore. thanks to the introduction of novel high-throughput technologies, it is now clear that there is not a direct correlation between the transcriptomes and the proteomes of cells or tissues. instead, mirnas, together with other ncrnas and rna binding proteins, play key roles in modulating the final gene output on its way to protein expression. this modulation, when combined with the abundance of apa detected in this study, suggests a more complex picture, where there are not only negative regulatory networks through mirnas, but also novel unexplored positive regulatory networks operated though apa. these positive networks are driven by genes that switch between 3’utr isoforms to escape mirna targeting, allowing their expression. in this view, both mirnas and apa can, in principle, dramatically reshape gene expression output, implying they both play key roles in the establishment and maintenance of cell and tissue identity. in this study we found that genes with tissue-specific 3’utr isoforms are enriched in microrna targets using both a three-species and a five-species conservation criteria. this was significantly more abundant than what we saw in randomly selected 3’utr isoforms using a three-species and a five-species conservation criteria in each tissue. we also found a similar enrichment comparing our dataset to the experimentally validated alg- footprints. our results in three worm somatic tissues link mirna regulation to apa, showing that microrna targets are much more abundant in ubiquitously expressed genes with tissue-specific 3’utr isoforms than in genes that do not use apa. recently, microrna populations from intestine and body muscle tissues were isolated using an rna-ip strategy, providing a tissue-level atlas of microrna expression. unfortunately, while this study suggests that mirnas are also pervasive in worm tissues, it is still unclear which genes they target, and further experiments have to be performed to highlight these regulatory networks. apaome.org: a resource for 3’utr biology we have compiled our tissue-specific transcriptomes into a useful online resource for scientists interested in 3’utr biology and apa. the apaome.org site uses an apache web server and several custom-made perl scripts that query a dedicated mysql database. it is currently hosted in the biodesign institute at arizona state university, and offers a simple and well-integrated interactive user interface to query gene records and 3’utr isoform data, giving access to a dedicated gbrowse installation specifically designed to study apa in worms. this database displays tracks for each tissue transcriptome, including tissue-specific apa, as well as curated 3’utr data from previously published studies.  here we present the first comparative analysis of the transcriptome, the 3’utrome, and the promoter diversity of three large somatic tissues of the soil nematode c. elegans. we found abundant tissue-specific gene expression changes that correlated with the presence of distinct promoter signatures between c. elegans intestine and muscle tissues, and defined thousands of tissue-specific splice isoforms. we have discovered that tissue-specific apa is pervasive in nematodes, and that 3’utr isoform changes correlated with gain or loss of mirna target elements, suggesting a role for apa in modulating tissue-specific post-transcriptional gene regulation. 
we have precisely mapped approximately tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3’utr isoforms significantly enriched with microrna targets. we pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to mirna regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells. the intestine transcriptome is expansive, expressing over 30% of c. elegans mrnas, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. tissue-specific rna plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. for the first time, pat-seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. here we developed a new method, polya-tagging and sequencing to isolate high-quality tissue-specific mrna from caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3’utromes. active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. we have identified thousands of novel genes and isoforms differentially expressed between these three tissues.
 tissue-specific rna plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. here we developed a new method, polya-tagging and sequencing to isolate high-quality tissue-specific mrna from caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3’utromes.  we have identified thousands of novel genes and isoforms differentially expressed between these three tissues. the intestine transcriptome is expansive, expressing over 30% of c. elegans mrnas, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. we have precisely mapped approximately tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3’utr isoforms significantly enriched with microrna targets.  for the first time, pat-seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. we pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to mirna regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rna-seqtissue-specific mrnaeleganspromotertranscriptionmrna-taggingtranscriptome3’utrmirnaspat-seqissue-copyright-statement© the author 2015 